Bispecific signaling agents and uses thereof

ABSTRACT

The present invention relates, in part, to bispecific chimeric proteins that find use in various immunotherapies based on various properties, including, for example, a dual immune cell recruitment and immune signal delivery function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Nos. 62/291,769, filed Feb. 5, 2016; 62/335,880, filed May13, 2016; 62/411,805, filed Oct. 24, 2016; 62/291,772, filed Feb. 5,2016; 62/291,774, filed Feb. 5, 2016; 62/335,965, filed May 13, 2016;62/291,776, filed Feb. 5, 2016; 62/335,968, filed May 13, 2016;62/335,979, filed May 13, 2016; 62/336,030, filed May 13, 2016,62/353,607, filed Jun. 23, 2016; and 62/291,779, filed Feb. 5, 2016, theentire contents of all of which are herein incorporated by reference.

FIELD

The present invention relates, in part, to chimeric proteins that canrecruit effector cells and deliver signaling to provide beneficialeffects.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:ORN-009PC_Sequence_listing; date recorded: Feb. 3, 2017; file size: 386KB).

BACKGROUND

Cancer is a global health challenge that causes nearly 7 million deathseach year worldwide and which has, to date, proven largely untreatabledespite major advances in medicine. Frustratingly, cancers appear todevelop strategies to evade immune detection and destruction therebysidestepping the body's main protection against disease.

Many recent immunotherapies have been developed to re-direct the body'simmune system towards cancers. Immunotherapy provides the advantage ofcell specificity that other treatment modalities, such as chemotherapyand radiation, lack. As such, methods for enhancing the efficacy ofimmune based therapies can be clinically beneficial. For example,costimulatory and coinhibitory molecules play a central role in theregulation of T cell immune responses. However, despite impressivepatient responses to agents targeting these costimulatory andcoinhibitory molecules, including, for example, clinical trials that ledto the approval of YERVOY, KEYTRUDA, and OPDIVO, checkpoint inhibitiontherapy still fails in the overwhelming majority of patients.

Further, most cancer treatments, including immunotherapies, requirecomplex regimens of various agents—each typically bringing a complexpattern of side effects that narrows a patient's therapeutic window fortreatment and makes the patient more susceptible to other diseases.

Therefore, there remains a need for improved immunotherapeutic agents,including those that can effectively modulate the immune system in amultifunctional manner.

SUMMARY

In some aspects, the present invention relates to chimeric proteinswhich find use in various targeted cell methods. In some aspects, thepresent invention relates to chimeric proteins having two or moretargeting moieties which have recognition domains (e.g. antigenrecognition domains, including without limitation various antibodyformats, inclusive of single-domain antibodies) which specifically bindto a target (e.g. antigen, receptor) of interest. The chimeric proteinfurther comprises a modified (e.g. mutant) signaling agent (forinstance, an immune-modulating agent), the modified (e.g. mutant)signaling agent having one or more modifications (e.g. mutations) thatprovide improved safety as compared to an unmodified (e.g. wild type)signaling agent. The targeting moieties and the modified (e.g. mutant)signaling agent are optionally connected by one or more linkers.

In various embodiments, the targeting moieties having recognitiondomains that specifically bind to a target (e.g. antigen, receptor) ofinterest, including those found on one or more immune cells, which caninclude, without limitation, T cells, cytotoxic T lymphocytes, T helpercells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g. M1 macrophages), neutrophils, B cells, anddendritic cells. In some embodiments, the recognition domainsspecifically bind to a target (e.g. antigen, receptor) of interest andeffectively recruit one of more immune cells. In some embodiments, thetargets (e.g. antigens, receptors) of interest can be found on one ormore tumor cells. In these embodiments, the present chimeric proteinsmay recruit an immune cell, e.g. an immune cell that can kill and/orsuppress a tumor cell, to a site of action (such as, by way ofnon-limiting example, the tumor microenvironment).

Furthermore, not only can the present chimeric proteins directly orindirectly recruit cells that increase the likelihood of a beneficialeffect, e.g. an anti-tumor effect, but also the present chimericproteins may deliver important signaling to the cells via signalingagents (e.g. via one or more of an interferon, interleukin, TNF, EPO,etc.). Further, such signaling agents may be mutated such that thesignaling is controlled and provides improved safety (e.g. does not comeat the cost of systemic toxicity). Further still, in variousembodiments, the present invention provides for various mutations to thesignaling agents that can, for example, temper activity at a site oftherapeutic action (e.g. at a therapeutic receptor) and also reduce oreliminate off-target binding at a non-therapeutic receptor. That is, insome embodiments, the signaling agents have reduced receptor promiscuityand controlled on-target action.

In various embodiments, the signaling agent can modulate one or morecells that are targeted by the targeting moieties (e.g. recruited cells,such as disease cells and/or effector cells). For instance, thesignaling agent can modulate one or both of the targeted cells (and thetargeted cells can be effector and/or disease cells), depending onwhether the targeted cells express a receptor for the signaling agent.

In various embodiments, the present chimeric proteins find use in thetreatment of various diseases or disorders such as cancer, infections,immune disorders, anemia, autoimmune diseases, cardiovascular diseases,wound healing, ischemia-related diseases, neurodegenerative diseases,metabolic diseases and many other diseases and disorders, and thepresent invention encompasses various methods of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, in panel A, C57BU6 mice were inoculated subcutaneously (50μl) with 6×10⁵ B16mCD20cl1 melanoma tumor cells. Perilesional treatmentwith 120 μg of an anti-PD-L1 VHH (120 μl) or a fusion of an anti-PD-L1VHH to human IFNalpha, Q124R mutant was started when tumors reached asize of ±10 mm² as measured by caliper. The curves are in the same order(top to bottom) as in the figure legend identifying the treatment types(e.g. PBS is the top curve, anti-PD-L1 is the middle curve, and thebottom curve is the fusion of an anti-PD-L1 VHH to human IFNalpha, Q124Rmutant). In panel B: C57BL/6 littermates were inoculated subcutaneously(50 μl) with 6×10⁵ B16mCD20cl1 melanoma tumor cells. Perilesionaltreatment with 30 μg of the indicated constructs (100 μl) was startedwhen tumors reached a size of ±10 mm² as measured by caliper. Blackarrows indicate the treatment-schedule. The curves are in the same order(top to bottom) as in the figure legend identifying the treatment types(e.g. top curve is PBS, middle curve is a fusion of an anti-CD20 VHH tohuman IFNalpha, Q124R mutant, and the bottom curve is a bi-specificconstruct: anti-CD20 VHH-human IFNalpha, Q124R mutant-anti-PD-L1 VHH).

FIG. 2 shows an experiment in which C57BL/6 mice were inoculatedsubcutaneously (50 μl) with 6×10⁵ B16mCD20cl1 melanoma tumor cells.Perilesional treatment with 30 μg of the indicated chimeras (100 μl) wasstarted when tumors reached a size of ±10 mm² as measured by caliper.Top row of arrows indicate the treatment-schedule. Bottom row of arrowsindicate perilesional injection of 120 μg of anti-PD-L1 VHH (120 μl).The curves are in the same order (top to bottom) as in the figure legendidentifying the treatment types. “PD-L1 Q124R” is a fusion of ananti-PD-L1 VHH to human IFNalpha, Q124R mutant; “Clec9A-Q124R” is afusion of an anti-Clec9A VHH to human IFNalpha, Q124R mutant; and“Clec9A-Q124R-PD-L1” is a bi-specific construct: anti-Clec9A VHH-humanIFNalpha, Q124R mutant-anti-PD-L1 VHH.

FIG. 3 shows various safety parameters of the bi-specificmCD20-Q124R-PD-L1 chimera. In panels B-G, the order of histograms is,left to right, PBS, “CD20-Q124R,” a fusion of an anti-CD20 VHH to humanIFNalpha Q124R mutant, and a bi-specific of anti-CD20 VHH-humanIFNalpha, Q124R mutant-anti-PD-L1 VHH (“CD20-Q124R-PD-L1”).

FIG. 4 shows various safety parameters of the bi-specificClec9A-Q124R-PD-L1 chimera. In panels B-G, the order of histograms is,left to right, PBS, anti-PD-L1, “Clec9A-Q124R,” a fusion of ananti-Clec9A VHH to human IFNalpha Q124R mutant, a bi-specific ofanti-Clec9A VHH-human IFNalpha, Q124R mutant-anti-PD-L1 VHH(“Clec9A-Q124R-PD-L1”), and “PD-L1-Q124R,” a fusion of an anti-PD-L1 VHHto human IFNalpha Q124R mutant.

FIG. 5 shows a mouse tumor growth study in which C57BU6 mice wereinoculated subcutaneously (50 μl) with 6×10⁵ B16 melanoma tumor cells.Perilesional treatment with 30 μg of the indicated treatment agents (100μl) was started when tumors reached a size of ±10 mm² as measured bycaliper. Graph shows the evolution of tumor size over the indicatedtime.

FIG. 6 shows a mouse tumor growth study in which C57BL/6 mice wereinoculated subcutaneously with B16 melanoma tumor cells. Perilesionaltreatment with the indicated treatment agents was started when tumorsreached certain size as measured by caliper. Graph shows the evolutionof tumor size over the indicated time.

FIG. 7 shows a mouse tumor growth study in which Batf3 knockout micethat lack cross-presenting dendritic cells were inoculatedsubcutaneously with B16 melanoma tumor cells. Perilesional treatmentwith the indicated treatment agents was started when tumors reachedcertain size as measured by caliper. Graph shows the evolution of tumorsize over the indicated time.

FIG. 8, panels A-C, show a mouse tumor growth study in which mice wereinoculated with 4T1 mammary tumor cells. The mice were treated with theindicated agents. Graphs show the evolution of tumor size over theindicated time.

FIG. 9 shows a mouse tumor growth study in which mice were inoculatedwith either B16 melanoma tumor cells (panel A) or 4T1 mammary tumorcells (panels B and C). The mice were then treated with the indicatedagents with or without doxorubicin. Graphs show the evolution of tumorsize over the indicated time.

FIG. 10 shows a mouse tumor growth study in which mice were inoculatedwith 4T1 mammary tumor cells. Tumor free mice from FIG. 9, panel B wererechallenged with 10⁵ 4 T1 cells, and tumor growth was monitored. Graphshows the evolution of tumor size over the indicated time.

FIG. 11 shows the efficacy of bispecific chimeras using an experimentalautoimmune encephalomyelitis (EAE) model. Panel A shows a schematic ofthe experimental design. Panel B shows the clinical score of micetreated with 100 IU of the indicated agents. Panel C shows the clinicalscore of mice treated with 1000 IU of the indicated agents. Panel Dshows that the Clec9A-CD20-hIFNa2-Q124R bispecific chimera was safe anddid not induce lymphopenia. The histogram bars represent, from left toright, treatment with PBS, a monospecific fusion of anti-Clec9A tomodified human IFN alpha Q124R, a monospecific fusion of anti-mCD20 tomodified human IFN alpha Q124R, a monospecific fusion of anti-Bcll10 tomodified human IFN alpha Q124R, wild type IFN, a combination of themonospecific fusion of anti-Clec9A to modified human IFN alpha Q124R andthe monospecific fusion of anti-mCD20 to modified human IFN alpha Q124R,the Clec9A-hIFNa2-Q124R-CD20 bispecific chimera, and theClec9A-CD20-hIFNa2-Q124R bispecific chimera. Panel E compares theefficacy of the Clec9A-hIFNa2-Q124R-CD20 bispecific chimera at 100 IUand 1000 IU dosages. Panel F compares the efficacy of theClec9A-CD20-hIFNa2-Q124R bispecific chimera at 100 IU and 1000 IUdosages.

FIG. 12 shows the efficacy of the Clec9A-CD20-hIFNa2-Q124R bispecificchimera at the 5000 IU dosage. Panel A shows the effect of thebispecific chimera on clinical score. Panel B shows the effect of thebispecific chimera on weight. Panel C shows the effect of the bispecificchimera on the incidence and onset of disease.

FIG. 13 shows an evaluation of tumor-antigen specific CD8⁺ T cellproliferation and activation both in tumor-draining lymph nodes (panelsA and B) as well as in spleen (panels C and D), two prime organs in theinduction of anti-tumor immunity. B16-OVA bearing mice were injectedonce with PBS, bispecific chimera of anti-Clec9A VHH/anti-PD-L1VHH/human IFN Q124R or the same bispecific chimera in combination withdoxorubicin one day after adoptive transfer of CFSE-labeled CD8⁺ OT-Icells carrying a CD8 TCR recognizing the model antigen ovalbumin (OVA),present on B16-OVA tumor cells. Proliferation and activation ofCFSE-labeled CD8⁺ OT-I cells was evaluated 4 days later by flowcytometry, showing enhanced proliferation and activation status of theproliferated cells in mice receiving the bispecific chimera ofanti-Clec9A VHH/anti-PD-L1 VHH/human IFN Q124R or the same bispecificchimera in combination with doxorubicin as compared to perilesionalinjection with PBS (panels A and C). Data show the percentage of OT-ICD8⁺ cells having undergone at least one division. One representativeflow cytometric profile is included. In addition, as shown in panels Band D, flow cytometric analysis of CD44 and CD62L expression wasperformed on CD8⁺ CFSE⁺ T cells showing activated and memory T cellphenotype upon delivery of bispecific chimera of anti-Clec9AVHH/anti-PD-L1 VHH/human IFN Q124R either alone or in combination withdoxorubicin. * P<0.05, P<0,01, ** P<0.001 and *** P<0.0001 compared withPBS treated animals by one-way ANOVA with Dunnett's multiple comparisontest.

FIG. 14 shows B16 cells stimulated with 100 ng/ml chimeras (or were leftunstimulated) and stained for phospho STAT1. Data are plotted as meanfluorescent intensities. Chimeras analyzed are: anti-Sirp1αVHH/anti-PD-L1 VHH/human IFN Q124R, anti-DNAM VHH/anti-PD-L1 VHH/humanIFN Q124R; and a monospecific fusion of anti-Bcll10 VHH to modifiedhuman IFN alpha Q124R (non-specific control).

FIG. 15 shows, in panels A, B, and C, human CD8 targeting ofmono-specific chimeras (anti-human CD8 VHH/human IFN R149A). Zebra-plotof CD8 versus pSTAT1 staining of stimulated PBMCs is shown in panel A.Panels B and C: mean fluorescent intensities (MFI) of pSTAT1 staining ofCD8-positive (panel B) or CD8-negative (panel C) cells are plotted. FIG.15 shows, in panels D, E, and F human CD8 targeting of bi-specificchimeras (anti-human CD8 VHH/anti-human PD-L1 VHH/human IFN R149A).

FIG. 16 shows an experiment in which MDA-MB-321 cells were stimulatedwith a serial dilution of chimeras and stained for phospho STAT1. Dataare plotted as mean fluorescent intensities (MFI). Chimeras studied wereanti-human PD-L1 VHH/human IFN R149A; anti-human PD-L1 VHH/human IFNR33A/E120R; anti-human Clec9A VHH/anti-human PD-L1 VHH/human IFN R149A;anti-human Clec9A VHH/anti-human PD-L1 VHH/human IFN R33A/E120R; andanti-human Bcl10 VHH/human IFN R149A. For reference, this is the orderof the constructs in FIG. 16, from top to bottom, when viewing the 4ng/ml point on the X axis.

FIG. 17 shows, Daudi, Jurkat and Wish cells were stimulated with aserial dilution scFv chimeras and stained for phospho STAT1. Data areplotted as mean fluorescent intensities (MFI). Chimeras studied wereanti-human CD20 scFv/anti-human CD3 scFv/IFN R149A. When viewing thegraphs at point 1000 on the x axis, the order of the curves is Jurkat,Daudi, and Wish cells.

FIG. 18 shows the antitumor effects of an anti-human Clec9aVHH/anti-human PDL1 VHH/human IFN-R149A bi-specific chimera on a humantumor (RL) grown in humanized mice. With reference to day 18 on the Xaxis, the constructs studied were, from top to bottom: PBS (control);anti-human Clec9a VHH/anti-human PDL1 VHH/human IFN-R149A bi-specificchimera; and anti-human Clec9a VHH/anti-human PDL1 VHH/human IFN-R149Abi-specific chimera plus FMS-like tyrosine kinase 3 ligand (FLT3L).

FIG. 19 shows a human dendritic cell pSTAT1 signaling assay. Chimerasstudied were anti-human Clec9A VHH/anti-human PD-L1 VHH/human IFN R149Aand anti-human Clec9A VHH/anti-human PD-L1 VHH/human IFN R33A/E120R. Twodoses of the agents were studied: 100 ng/ml and 500 ng/ml. PBS was thecontrol and the data are expressed as a fold change of the percentage ofpSTAT1⁺ dendritic cells (data is an average of a triplicate data set).

DETAILED DESCRIPTION

The present invention is based, in part, on the surprising discoverythat bi-specific chimeric proteins with signaling agents bearingtherapeutically beneficial mutations provide beneficial therapeuticproperties and reduced side effects. In various embodiments, thesechimeric proteins may recruit the appropriate cells (e.g. immune cells)to site of in need of therapeutic action (e.g. a tumor cell) via thedual targeting moieties and, in addition, delivery one or more importantsignals to effect a therapeutic signal (e.g. via one or more modifiedsignaling agents). The present invention provides pharmaceuticalcompositions comprising the chimeric proteins and their use in thetreatment of various diseases. Administration of the chimeric proteinsand pharmaceutical compositions of the invention achieves significantlyreduced side effects compared to the wild type soluble agent.

Chimeric Proteins

In various embodiments, the present invention relates to a bi-specificor multifunctional chimeric protein having two or more targetingmoieties having recognition domains that specifically bind to a target(e.g. antigen, receptor) of interest, which optionally, and directly orindirectly, recruit cells of relevance and/or modulate the function ofthe recruited cells, and a modified signaling agent, which bears one ormore mutations that render the signaling agent suitable forpharmaceutical use with minimal side effects (e.g. minimal cytokinestorm-like effects, flu-like symptoms, suicidal thoughts, off-targetside effects, among others).

In various embodiments, the chimeric protein, among other features,directly or indirectly recruits one or more effector cells to a diseasecell, e.g. via the targeting moieties, and further delivers a signal tothe effector cell to modulate the disease cell in atherapeutically-relevant manner. In various embodiments, the chimericprotein, among other features, directly or indirectly recruits one ormore effector cells to a disease cell, e.g. via the targeting moieties,and further delivers a signal to the disease cell to modulate theeffector cell in a therapeutically-relevant manner. In variousembodiments, the chimeric protein, among other features, directly orindirectly recruits one or more effector cells to a disease cell, e.g.via the targeting moieties, and further delivers a signal to theeffector cell to modulate the effector cell in atherapeutically-relevant manner. In various embodiments, the chimericprotein, among other features, directly or indirectly recruits one ormore effector cells to a disease cell, e.g. via the targeting moieties,and further delivers a signal to the disease cell to modulate thedisease cell in a therapeutically-relevant manner. In some embodiments,the signaling agent effects effector cell and/or the disease cell.

In various embodiments, the present chimeric protein provides at leasttwo therapeutic benefits when used pharmaceutically. For instance, thepresent chimeric protein may effectively recruit proper cells to a siteof required therapy (by way of non-limiting example, immune effectorcells to a tumor, e.g. the tumor microenvironment) and deliver one ormore signals to the cells—e.g. immune effector cells and/or tumorcells—to promote a cancer reducing or eliminating effect (e.g. provideimmune cell stimulation from the signaling agent, provide immuneco-stimulatory signals via the targeting domains, provide reduction orsilencing of immune co-inhibitory signals via the targeting domains,etc.). Accordingly, as described herein, the present chimeric proteinprovides a platform of therapeutically-relevant options for theeffective treatment of diseases via the immune system, including,without limitation, the treatment of cancer.

Furthermore, the present chimeric protein, in various embodiments, haspharmacodynamic and pharmacokinetic properties that make it particularlysuited for use in therapies. For example, in various embodiments,including embodiments in which smaller antibody-based formats are usedfor targeting (as described elsewhere herein), the present chimericprotein has a molecular weight that allows avoidance of renal excretionto allow for therapeutic effects at a low dose (e.g. without loss of thetherapeutic via the kidney). For instance, the present chimeric protein,in various embodiments, is engineered to have a molecular mass of about50 KDa, or about 60 KDa, or about 70 KDa, or about 80 KDa, or about 90KDa, or about 100 KDa. In various embodiments, the present constructsevade kidney filtration such that they do not require external methodsof half-life extension.

In various embodiments, each of the individual chimeric proteins may byconjugated and/or fused with another agent to extend half-life orotherwise improve pharmacodynamic and pharmacokinetic properties. Insome embodiments, the chimeric proteins may be fused or conjugated withone or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN),albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP),PAS, HAP, GLK, CTP, transferrin, and the like. In some embodiments, thechimeric protein may be fused or conjugated with an antibody or anantibody fragment such as an Fc fragment. For example, the chimericprotein may be fused to either the N-terminus or the C-terminus of theFc domain of human immunoglobulin (Ig) G. In various embodiments, eachof the individual chimeric proteins is fused to one or more of theagents described in BioDrugs (2015) 29:215-239, the entire contents ofwhich are hereby incorporated by reference.

As described herein, the present chimeric protein may have improvedsafety due to one of more modifications, e.g. mutations. In variousembodiments, improved safety means that the present chimeric proteinprovides lower toxicity (e.g. systemic toxicity and/ortissue/organ-associated toxicities); and/or lessened or substantiallyeliminated side effects; and/or increased tolerability, lessened orsubstantially eliminated adverse events; and/or reduced or substantiallyeliminated off-target effects; and/or an increased therapeutic window.

In some embodiments, the present chimeric protein increases thetherapeutic index (e.g., by about 2-fold, about 5-fold, about 10-fold,about 25-fold, about 50-fold, about 100-fold, about 200-fold, or more)of any protein therapeutic (e.g., based on a naturally occurring orengineered protein or fragment thereof) by reducing the general toxicityof the protein and maintaining or increasing the selective effect of theprotein on target cells or tissues. For instance, this increase intherapeutic index (e.g., by about 2-fold, about 5-fold, about 10-fold,about 25-fold, about 50-fold, about 100-fold, about 200-fold, or more)may be of the modified signaling agent (e.g. relative to the wild typesignaling agent, or relative to a wild type signaling agent in thecontext of a fusion protein with a single targeting moiety, or relativeto a wild type signaling agent in the context of a fusion protein withmore than one targeting moieties).

In some embodiments, the present chimeric protein allows for efficientbinding of the targeting moieties and the signaling agent to theirreceptors. For instance, in some embodiments, the chimeric proteinallows for efficient binding of one of the targeting moieties and thesignaling agent to receptors on the same cell (e.g., differentreceptors) as well as the efficient binding of the other targetingmoiety to another cell. As described elsewhere herein, in variousembodiments, the signaling agent is mutated to provide attenuatedactivity, and the binding of the targeting moiety and the signalingagent to receptors on the same cell is sequential, e.g. targetingmoiety/receptor binding preceding signaling agent/receptor binding. Forinstance, in some embodiments the signaling agent by itself issignificantly less active in its mutated form (e.g. relative to wildtype) because it cannot efficiently bind to its receptor(s).Accordingly, chimeric proteins of the invention are useful to avoidunwanted side effects caused by the signaling agent binding to itsnatural receptor on non-target cells. However, the signaling agent isactive on target cells because the targeting moiety(ies) compensates forthe missing/insufficient binding (e.g., without limitation and/oravidity) required for substantial activation. In various embodiments,the chimeric proteins of the present invention have a modified (e.g.mutant) signaling agent which causes the signaling agent to be inactiveen route to the site of therapeutic activity (e.g. in contact with atarget cell, including a tumor cell) through the body and to have itseffect substantially on specifically targeted cell types which greatlyreduces undesired side effects

In various embodiments, the present chimeric proteins have selectivebioactivity, e.g. therapeutically-relevant bioactivity, towards targetedcells (e.g. tumor cells), but not towards cells that are not targeted(e.g. normal, non-tumor cells).

In various embodiments, the present chimeric proteins providesynergistic activity and/or therapeutic effects. In such embodiments,the activity and/or therapeutic effects of the chimeric proteins haveimproved therapeutic effects, e.g. synergistically greater, than thetherapeutic effects of the individual components (i.e., the targetingmoieties and the signaling agent) administered alone or in combinationvia co-administration.

In various embodiments, the present chimeric proteins have two or moretargeting moieties and the signaling agent connected together viadifferent configurations. In an embodiment, one of the targetingmoieties is linked to the amino-terminus of the signaling agent and theother targeting moiety is linked to the carboxy-terminus of thesignaling agent. In another embodiment, both targeting moieties arelinked to the amino-terminus of the signaling agent. For example, theamino-terminus of the signaling agent may be linked to thecarboxy-terminus of one of the targeting moieties, which in turn islinked to the other targeting moiety (e.g., via its amino-terminus). Ina further embodiment, both targeting moieties are linked to thecarboxy-terminus of the signaling agent. For example, thecarboxy-terminus of the signaling agent may be linked to theamino-terminus of one of the targeting moieties, which in turn is linkedto the other targeting moiety (e.g., via its carboxy-terminus).

In various embodiments, the present chimeric proteins are in the form ofa fusion protein having the components described herein.

In various embodiments, the present chimeric proteins have two or moretargeting moieties that target different antigens or receptors, and onetargeting moiety may be attenuated for its antigen or receptor, e.g. thetargeting moiety binds its antigen or receptor with a low affinity oravidity (including, for example, at an affinity or avidity that is lessthan the affinity or avidity the other targeting moiety has for its forits antigen or receptor, for instance the difference between the bindingaffinities may be about 10-fold, or 25-fold, or 50-fold, or 100-fold, or300-fold, or 500-fold, or 1000-fold, or 5000-fold; for instance thelower affinity or avidity targeting moiety may bind its antigen orreceptor at a K_(D) in the mid- to high-nM or low- to mid-μM range whilethe higher affinity or avidity targeting moiety may bind its antigen orreceptor at a K_(D) in the mid- to high-pM or low- to mid-nM range). Forinstance, in some embodiments, the present chimera comprises anattenuated targeting moiety that is directed against a promiscuousantigen or receptor, which may improve targeting to a cell of interest(e.g. via the other targeting moiety) and prevent effects acrossmultiple types of cells, including those not being targeted for therapy(e.g. by binding promiscuous antigen or receptor at a higher affinitythan what is provided in these embodiments).

In various embodiments, the present invention provides for an isolatednucleic acid encoding a chimeric protein as described herein.

Targeting Moiety Cellular Recruitment

In various embodiments, the chimeric proteins of the present inventionhave targeting moieties having recognition domains which specificallybind to a target (e.g. antigen, receptor) of interest. In variousembodiments, the chimeric proteins of the present invention have one ormore targeting moieties which target different cells (e.g. to make asynapse) or the same cell (e.g. to get a more concentrated signalingagent or therapeutic effect). The target (e.g. antigen, receptor) ofinterest can be found on one or more immune cells, which can include,without limitation, T cells, cytotoxic T lymphocytes, T helper cells,natural killer (NK) cells, natural killer T (NKT) cells, anti-tumormacrophages (e.g. M1 macrophages), B cells, dendritic cells, or subsetsthereof. In some embodiments, the recognition domains specifically bindto a target (e.g. antigen, receptor) of interest and effectively,directly or indirectly, recruit one of more immune cells. In someembodiments, the target (e.g. antigen, receptor) of interest can befound on one or more tumor cells. In these embodiments, the presentchimeric proteins may directly or indirectly recruit an immune cell,e.g., in some embodiments, to a therapeutic site (e.g. a locus with oneor more disease cell or cell to be modulated for a therapeutic effect).In some embodiments, the present chimeric proteins may directly orindirectly recruit an immune cell, e.g. an immune cell that can killand/or suppress a tumor cell, to a site of action (such as, by way ofnon-limiting example, the tumor microenvironment).

In various embodiments, the chimeric proteins have targeting moietieshaving recognition domains which specifically bind to a target (e.g.antigen, receptor) which is part of a non-cellular structure. In someembodiments, the antigen or receptor is not an integral component of anintact cell or cellular structure. In some embodiments, the antigen orreceptor is an extracellular antigen or receptor. In some embodiments,the target is a non-proteinaceous, non-cellular marker, including,without limitation, nucleic acids, inclusive of DNA or RNA, such as, forexample, DNA released from necrotic tumor cells or extracellulardeposits such as cholesterol.

In some embodiments, the target (e.g. antigen, receptor) of interest ispart of the non-cellular component of the stroma or the extracellularmatrix (ECM) or the markers associated therewith. As used herein, stromarefers to the connective and supportive framework of a tissue or organ.Stroma may include a compilation of cells such asfibroblasts/myofibroblasts, glial, epithelia, fat, immune, vascular,smooth muscle, and immune cells along with the extracellular matrix(ECM) and extracellular molecules. In various embodiments, the target(e.g. antigen, receptor) of interest is part of the non-cellularcomponent of the stroma such as the extracellular matrix andextracellular molecules. As used herein, the ECM refers to thenon-cellular components present within all tissues and organs. The ECMis composed of a large collection of biochemically distinct componentsincluding, without limitation, proteins, glycoproteins, proteoglycans,and polysaccharides. These components of the ECM are usually produced byadjacent cells and secreted into the ECM via exocytosis. Once secreted,the ECM components often aggregate to form a complex network ofmacromolecules. In various embodiments, the chimeric protein of theinvention comprises a targeting moiety that recognizes a target (e.g.,an antigen or receptor or non-proteinaceous molecule) located on anycomponent of the ECM. Illustrative components of the ECM include,without limitation, the proteoglycans, the non-proteoglycanpolysaccharides, fibers, and other ECM proteins or ECM non-proteins,e.g. polysaccharides and/or lipids, or ECM associated molecules (e.g.proteins or non-proteins, e.g. polysaccharides, nucleic acids and/orlipids).

In some embodiments, the targeting moiety recognizes a target (e.g.antigen, receptor) on ECM proteoglycans. Proteoglycans are glycosylatedproteins. The basic proteoglycan unit includes a core protein with oneor more covalently attached glycosaminoglycan (GAG) chains.Proteoglycans have a net negative charge that attracts positivelycharged sodium ions (Na+), which attracts water molecules via osmosis,keeping the ECM and resident cells hydrated. Proteoglycans may also helpto trap and store growth factors within the ECM. Illustrativeproteoglycans that may be targeted by the chimeric proteins of theinvention include, but are not limited to, heparan sulfate, chondroitinsulfate, and keratan sulfate. In an embodiment, the targeting moietyrecognizes a target (e.g. antigen, receptor) on non-proteoglycanpolysaccharides such as hyaluronic acid.

In some embodiments, the targeting moiety recognizes a target (e.g.antigen, receptor) on ECM fibers. ECM fibers include collagen fibers andelastin fibers. In some embodiments, the targeting moiety recognizes oneor more epitopes on collagens or collagen fibers. Collagens are the mostabundant proteins in the ECM. Collagens are present in the ECM asfibrillar proteins and provide structural support to resident cells. Inone or more embodiments, the targeting moiety recognizes and binds tovarious types of collagens present within the ECM including, withoutlimitation, fibrillar collagens (types I, II, III, V, XI), facitcollagens (types IX, XII, XIV), short chain collagens (types VIII, X),basement membrane collagens (type IV), and/or collagen types VI, VII, orXIII. Elastin fibers provide elasticity to tissues, allowing them tostretch when needed and then return to their original state. In someembodiments, the target moiety recognizes one or more epitopes onelastins or elastin fibers.

In some embodiments, the targeting moiety recognizes one or more ECMproteins including, but not limited to, a tenascin, a fibronectin, afibrin, a laminin, or a nidogen/entactin.

In an embodiment, the targeting moiety recognizes and binds to tenascin.The tenascin (TN) family of glycoproteins includes at least fourmembers, tenascin-C, tenascin-R, tenascin-X, and tenascin W. The primarystructures of tenascin proteins include several common motifs ordered inthe same consecutive sequence: amino-terminal heptad repeats, epidermalgrowth factor (EGF)-like repeats, fibronectin type III domain repeats,and a carboxyl-terminal fibrinogen-like globular domain. Each proteinmember is associated with typical variations in the number and nature ofEGF-like and fibronectin type III repeats. Isoform variants also existparticularly with respect to tenascin-C. Over 27 splice variants and/orisoforms of tenascin-C are known. In a particular embodiment, thetargeting moiety recognizes and binds to tenascin-CA1. Similarly,tenascin-R also has various splice variants and isoforms. Tenascin-Rusually exists as dimers or trimers. Tenascin-X is the largest member ofthe tenascin family and is known to exist as trimers. Tenascin-W existsas trimers. In some embodiments, the targeting moiety recognizes one ormore epitopes on a tenascin protein. In some embodiments, the targetingmoiety recognizes the monomeric and/or the dimeric and/or the trimericand/or the hexameric forms of a tenascin protein.

In an embodiment, the targeting moieties recognize and bind tofibronectin. Fibronectins are glycoproteins that connect cells withcollagen fibers in the ECM, allowing cells to move through the ECM. Uponbinding to integrins, fibronectins unfolds to form functional dimers. Insome embodiments, the targeting moiety recognizes the monomeric and/orthe dimeric forms of fibronectin. In some embodiments, the targetingmoiety recognizes one or more epitopes on fibronectin. In illustrativeembodiments, the targeting moiety recognizes fibronectin extracellulardomain A (EDA) or fibronectin extracellular domain B (EDB). Elevatedlevels of EDA are associated with various diseases and disordersincluding psoriasis, rheumatoid arthritis, diabetes, and cancer. In someembodiments, the targeting moiety recognizes fibronectin that containsthe EDA isoform and may be utilized to target the chimeric protein todiseased cells including cancer cells. In some embodiments, thetargeting moiety recognizes fibronectin that contains the EDB isoform.In various embodiments, such targeting moieties may be utilized totarget the chimeric protein to tumor cells including the tumorneovasculature.

In an embodiment, the targeting moiety recognizes and binds to fibrin.Fibrin is another protein substance often found in the matrix network ofthe ECM. Fibrin is formed by the action of the protease thrombin onfibrinogen which causes the fibrin to polymerize. In some embodiments,the targeting moiety recognizes one or more epitopes on fibrin. In someembodiments, the targeting moiety recognizes the monomeric as well asthe polymerized forms of fibrin.

In an embodiment, the targeting moiety recognizes and binds to laminin.Laminin is a major component of the basal lamina, which is a proteinnetwork foundation for cells and organs. Laminins are heterotrimericproteins that contain an α-chain, a β-chain, and a γ-chain. In someembodiments, the targeting moiety recognizes one or more epitopes onlaminin. In some embodiments, the targeting moiety recognizes themonomeric, the dimeric as well as the trimeric forms of laminin.

In an embodiment, the targeting moiety recognizes and binds to a nidogenor entactin. Nidogens/entactins are a family of highly conserved,sulfated glycoproteins. They make up the major structural component ofthe basement membranes and function to link laminin and collagen IVnetworks in basement membranes. Members of this family include nidogen-1and nidogen-2. In various embodiments, the targeting moiety recognizesan epitope on nidogen-1 and/or nidogen-2.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes an epitope present on any of thetargets (e.g., ECM proteins) described herein. In an embodiment, theantigen-recognition domain recognizes one or more linear epitopespresent on the protein. As used herein, a linear epitope refers to anycontinuous sequence of amino acids present on the protein. In anotherembodiment, the antigen-recognition domain recognizes one or moreconformational epitopes present on the protein. As used herein, aconformation epitope refers to one or more sections of amino acids(which may be discontinuous) which form a three-dimensional surface withfeatures and/or shapes and/or tertiary structures capable of beingrecognized by an antigen recognition domain.

In various embodiments, the targeting moiety may bind to the full-lengthand/or mature forms and/or isoforms and/or splice variants and/orfragments and/or any other naturally occurring or synthetic analogs,variants, or mutants of any of the targets (e.g., ECM proteins)described herein. In various embodiments, the targeting moiety may bindto any forms of the proteins described herein, including monomeric,dimeric, trimeric, tetrameric, heterodimeric, multimeric and associatedforms. In various embodiments, the targeting moiety may bind to anypost-translationally modified forms of the proteins described herein,such as glycosylated and/or phosphorylated forms.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes extracellular molecules such as DNA.In some embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes DNA. In an embodiment, the DNA isshed into the extracellular space from necrotic or apoptotic tumor cellsor other diseased cells.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes one or more non-cellular structuresassociated with atherosclerotic plaques. Two types of atheroscleroticplaques are known. The fibro-lipid (fibro-fatty) plaque is characterizedby an accumulation of lipid-laden cells underneath the intima of thearteries. Beneath the endothelium there is a fibrous cap covering theatheromatous core of the plaque. The core includes lipid-laden cells(macrophages and smooth muscle cells) with elevated tissue cholesteroland cholesterol ester content, fibrin, proteoglycans, collagen, elastin,and cellular debris. In advanced plaques, the central core of the plaqueusually contains extracellular cholesterol deposits (released from deadcells), which form areas of cholesterol crystals with empty, needle-likeclefts. At the periphery of the plaque are younger foamy cells andcapillaries. A fibrous plaque is also localized under the intima, withinthe wall of the artery resulting in thickening and expansion of the walland, sometimes, spotty localized narrowing of the lumen with someatrophy of the muscular layer. The fibrous plaque contains collagenfibers (eosinophilic), precipitates of calcium (hematoxylinophilic) andlipid-laden cells. In some embodiments, the targeting moiety recognizesand binds to one or more of the non-cellular components of these plaquessuch as the fibrin, proteoglycans, collagen, elastin, cellular debris,and calcium or other mineral deposits or precipitates. In someembodiments, the cellular debris is a nucleic acid, e.g. DNA or RNA,released from dead cells.

In various embodiments, the targeting moiety comprises an antigenrecognition domain that recognizes one or more non-cellular structuresfound in the brain plaques associated with neurodegenerative diseases.In some embodiments, the targeting moiety recognizes and binds to one ormore non-cellular structures located in the amyloid plaques found in thebrains of patients with Alzheimer's disease. For example, the targetingmoiety may recognize and bind to the peptide amyloid beta, which is amajor component of the amyloid plaques. In some embodiments, thetargeting moiety recognizes and binds to one or more non-cellularstructures located in the brains plaques found in patients withHuntington's disease. In various embodiments, the targeting moietyrecognizes and binds to one or more non-cellular structures found inplaques associated with other neurodegenerative or musculoskeletaldiseases such as Lewy body dementia and inclusion body myositis.

In some embodiments, the chimeric proteins of the invention may have twoor more targeting moieties that bind to non-cellular structures. In someembodiments, there are two targeting moieties and one targets a cellwhile the other targets a non-cellular structure. In variousembodiments, the targeting moieties can directly or indirectly recruitcells, such as disease cells and/or effector cells. In variousembodiments, the signaling agent can modulate one or more cells that aretargeted by the targeting moieties (e.g. recruited cells, such asdisease cells and/or effector cells). For instance, the signaling agentcan modulate one or both of the targeted cells (and the targeted cellscan be effector and/or disease cells), depending on whether the targetedcells express a receptor for the signaling agent.

In some embodiments, the present chimeric proteins are capable of, orfind use in methods involving, shifting the balance of immune cells infavor of immune attack of a tumor. For instance, the present chimericproteins can shift the ratio of immune cells at a site of clinicalimportance in favor of cells that can kill and/or suppress a tumor (e.g.T cells, cytotoxic T lymphocytes, T helper cells, natural killer (NK)cells, natural killer T (NKT) cells, anti-tumor macrophages (e.g. M1macrophages), B cells, dendritic cells, or subsets thereof) and inopposition to cells that protect tumors (e.g. myeloid-derived suppressorcells (MDSCs), regulatory T cells (Tregs); tumor associated neutrophils(TANs), M2 macrophages, tumor associated macrophages (TAMs), or subsetsthereof). In some embodiments, the present chimeric protein is capableof increasing a ratio of effector T cells to regulatory T cells.

For example, in some embodiments, the targeting moiety comprises anantigen recognition domain that specifically binds to a target (e.g.antigen, receptor) associated with T cells. In some embodiments, thetargeting moiety comprises an antigen recognition domain that directlyor indirectly recruits T cells. In an embodiment, the targeting moietycomprises an antigen recognition domain that specifically binds toeffector T cells. In some embodiments, the targeting moiety comprises anantigen recognition domain that directly or indirectly recruits effectorT cells, e.g., in some embodiments, to a therapeutic site (e.g. a locuswith one or more disease cell or cell to be modulated for a therapeuticeffect). Illustrative effector T cells include cytotoxic T cells (e.g.αβ TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺ effector T cells (e.g. αβ TCR, CD3⁺,CD4⁺, CCR7⁺, CD62Lhi, IL-7R/CD127⁺); CD8⁺ effector T cells (e.g. αβ TCR,CD3⁺, CD8⁺, CCR7⁺, CD62Lhi, IL-7RCD127⁺); effector memory T cells (e.g.CD62Llow, CD44⁺, TCR, CD3⁺, IL-7R/CD127⁺, IL-15R⁺, CCR7low); centralmemory T cells (e.g. CCR7⁺, CD62L⁺, CD27⁺, or CCR7hi, CD44⁺, CD62Lhi,TCR, CD3⁺, IL-7RCD127⁺, IL-15R⁺); CD62L⁺ effector T cells; CD8⁺ effectormemory T cells (TEM) including early effector memory T cells (CD27⁺CD62L⁻) and late effector memory T cells (CD27⁻ CD62L⁻) (TemE and TemL,respectively); CD127(⁺)CD25(low/−) effector T cells; CD127(⁻)CD25(⁻)effector T cells; CD8⁺ stem cell memory effector cells (TSCM) (e.g.CD44(low)CD62L(high)CD122(high)sca(⁺)); TH1 effector T-cells (e.g.CXCR3⁺, CXCR6⁺ and CCR5⁺, or αβ TCR, CD3⁺, CD4⁺, IL-12R⁺, IFNγR⁺,CXCR3⁺), TH2 effector T cels (e.g. CCR3⁺, CCR4⁺ and CCR8⁺, or αβ TCR,CD3⁺, CD4⁺, IL-4R⁺, IL-33R⁺, CCR4⁺, IL-17RB⁺, CRTH2⁺); TH9 effector Tcells (e.g. αβ TCR, CD3⁺, CD4⁺); TH17 effector T cells (e.g. αβ TCR,CD3⁺, CD4⁺, IL-23R⁺, CCR6⁺, IL-1R⁺); CD4⁺CD45RO⁺CCR7⁺ effector T cells,ICOS⁺ effector T cells; CD4⁺CD45RO⁺CCR7(⁻) effector T cells; andeffector T cells secreting IL-2, IL-4 and/or IFN-γ.

Illustrative T cell antigens of interest include, for example (andinclusive of the extracellular domains, where applicable): CD8, CD3,SLAMF4, IL-2Rα, 4-1BB/TNFRSF9, IL-2 R β, ALCAM, B7-1, IL-4 R, B7-H3,BLAME/SLAMFS, CEACAM1, IL-6 R, CCR3, IL-7 Rα, CCR4, CXCRI/IL-S RA, CCR5,CCR6, IL-10R α, CCR 7, IL-I0 R β, CCRS, IL-12 R β1, CCR9, IL-12 R β2,CD2, IL-13 R α 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d,ILT5/CDS5a, lutegrin α 4/CD49d, CDS, Integrin α E/CD103, CD6, Integrin αM/CD 11 b, CDS, Integrin α X/CD11c, Integrin β 2/CDIS, KIR/CD15S,CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd,CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45,LAJR2, CDS3, Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97,NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6,Common γ ChainML-2 R γ, Osteopontin, CRACC/SLAMF7, SLAMF7 (CS1), PD-1,CRTAM, PSGL-1, CTLA-4, RANKITNFRSF11A, CX3CR1, CX3CL1, L-Selectin,CXCR3, SIRP β 1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin,EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, FasLigand/TNFSF6, TIM-4, Fcγ RIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin,TNF RIII/TNFRSF1B, TRAIL RI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B,ICAM-2/CD102, TRAILR3/TNFRSF10C, IFN-γR1, TRAILR4/TNFRSF100, IFN-γ R2,TSLP, IL-1 R1 and TSLP R. In various embodiments, a targeting moiety ofthe chimeric protein binds one or more of these illustrative T cellantigens.

In an exemplary embodiment, the present chimeric protein comprises atargeting moiety directed against CD8. In various embodiments, thetargeting moiety directed against CD8 is a protein-based agent capableof specific binding to CD8 without functional modulation (e.g. partialor complete neutralization) of CD8. CD8 is a heterodimeric type Itransmembrane glycoprotein, whose α and β chains are both composed of animmunoglobulin (Ig)-like extracellular domain connected by an extendedO-glycosylated stalk to a single-pass transmembrane domain and a shortcytoplasmic tail (Li et al., 2013). The cytoplasmic region of theα-chain contains two cysteine motifs that serve as a docking site forsrc tyrosine kinase p561ck (Lck). In contrast, this Lck binding domainappears to be absent from the β chain, suggesting that the CD8 β chainis not involved in downstream signaling (Artyomov et al., 2010). CD8functions as a co-receptor for the T-cell receptor with its principlerole being the recruitment of Lck to the TCR-pMHC complex followingco-receptor binding to MHC (Turner et al., 1990, Veillette et al.,1988). The increase in the local concentration of this kinase activatesa signaling cascade that recruits and activates-chain-associated proteinkinase 70 (ZAP-70), subsequently leading to the amplification orenhancement of T-cell activation signals (Purbhoo et al., 2001, Laugelet al., 2007a).

In various embodiments, the chimeric protein of the invention comprisesa targeting moiety having an antigen recognition domain that recognizesan epitope present on the CD8 α and/or β chains. In an embodiment, theantigen-recognition domain recognizes one or more linear epitopes on theCD8 α and/or β chains. As used herein, a linear epitope refers to anycontinuous sequence of amino acids present on the CD8 α and/or β chains.In another embodiment, the antigen-recognition domain recognizes one ormore conformational epitopes present on the CD8 α and/or β chains. Asused herein, a conformation epitope refers to one or more sections ofamino acids (which may be discontinuous) which form a three-dimensionalsurface with features and/or shapes and/or tertiary structures capableof being recognized by an antigen recognition domain.

In various embodiments, the present chimeric protein may comprise atargeting moiety that may bind to the full-length and/or mature formsand/or isoforms and/or splice variants and/or fragments and/or any othernaturally occurring or synthetic analogs, variants, or mutants of humanCD8 α and/or β chains. In various embodiments, the targeting moietydirected against CD8 may bind to any forms of the human CD8 α and/or βchains, including monomeric, dimeric, heterodimeric, multimeric andassociated forms. In an embodiment, the targeting moiety directedagainst CD8 may bind to the monomeric form of CD8 α chain or CD8 βchain. In another embodiment, the targeting moiety directed against CD8may bind to a homodimeric form comprised of two CD8 α chains or two CD8β chains. In a further embodiment, the targeting moiety directed againstCD8 may bind to a heterodimeric form comprised of one CD8 α chain andone CD8 β chain.

In an embodiment, the present chimeric protein comprises a targetingmoiety with an antigen recognition domain that recognizes one or moreepitopes present on the human CD8 α chain. In an embodiment, the humanCD8 α chain comprises the amino acid sequence of:

Isoform 1 (SEQ ID NO: 1)MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV.

In an embodiment, the human CD8 α chain comprises the amino acidsequence of:

Isoform 2 (SEQ ID NO: 2)MALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAGNRRRVCKCPRPVVKSGDKPSLSARYV.

In an embodiment, the human CD8 α chain comprises the amino acidsequence of:

Isoform 3 (SEQ ID NO: 3)MRNQAPGRPKGATFPPRRPTGSRAPPLAPELRAKQRPGERVMALPVTALLLPLALLLHAARPSQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQNKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPWKSGDKPSLSARYV.

In an embodiment, the present chimeric protein comprises a targetingmoiety with an antigen recognition domain that recognizes one or moreepitopes present on the human CD8 β chain. In an embodiment, the humanCD8 β chain comprises the amino acid sequence of:

Isoform 1 (SEQ ID NO: 4)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRA RLRFMKQFYK.

In an embodiment, the human CD8 β chain comprises the amino acidsequence of:

Isoform 2 (SEQ ID NO: 5)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQLRLHPLEKCSRMDY.

In an embodiment, the human CD8 β chain comprises the amino acidsequence of:

Isoform 3 (SEQ ID NO: 6)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGRRRRARLRFMKQPQGEGISGTFVPQCLHGYYSNTT TSQKLLNPWILKT.

In an embodiment, the human CD8 β chain comprises the amino acidsequence of:

Isoform 4 (SEQ ID NO: 7)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQKFNIVCLKISGFTTCCCFQILQISREYGFGVLLQKDIGQ.

In an embodiment, the human CD8 β chain comprises the amino acidsequence of:

Isoform 5 (SEQ ID NO: 8)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLLVSLGVAIHLCCRRRRARLRFMKQPQGEGISGTFVPQCLHGYYSNTTTSQKLLNPWILKT.

In an embodiment, the human CD8 β chain comprises the amino acidsequence of:

Isoform 6 (SEQ ID NO: 9)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGRRRRARLRFMKQFYK.

In an embodiment, the human CD8 β chain comprises the amino acidsequence of:

Isoform 7 (SEQ ID NO: 10)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKDFTNKQRIGFWCPATKRHRSVMSTMWKNERRDTFNP GEFNGC.

In an embodiment, the human CD8 β chain comprises the amino acidsequence of:

Isoform 8 (SEQ ID NO: 11)MRPRLWLLLAAQLTVLHGNSVLQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWDSAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGTQLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGLKGKVYQEPLSPNACMDTTAILQPHRSCLTHGS.

In various embodiments, the present chimeric protein comprises atargeting moiety capable of specific binding. In various embodiments,the chimeric protein comprises a targeting moiety having an antigenrecognition domain such as an antibody or derivatives thereof. In anembodiment, the CD8 binding agent comprises a targeting moiety which isan antibody. In various embodiments, the antibody is a full-lengthmultimeric protein that includes two heavy chains and two light chainsas described elsewhere herein. In some embodiments, the antibody is achimeric antibody. In some embodiments, the antibody is a humanizedantibody.

In some embodiments, the present chimeric protein comprises a targetingmoiety directed against CD8 which is a single-domain antibody, such as aVHH. The VHH may be derived from, for example, an organism that producesVHH antibody such as a camelid, a shark, or the VHH may be a designedVHH. VHHs are antibody-derived therapeutic proteins that contain theunique structural and functional properties of naturally-occurringheavy-chain antibodies. VHH technology is based on fully functionalantibodies from camelids that lack light chains. These heavy-chainantibodies contain a single variable domain (V_(H)H) and two constantdomains (CH2 and CH3). VHHs are commercially available under thetrademark of NANOBODY or NANOBODIES. In an embodiment, the presentchimeric protein comprises a VHH.

In some embodiments, the present chimeric protein comprises a targetingmoiety directed against CD8 which is a VHH comprising a single aminoacid chain having four “framework regions” or FRs and three“complementary determining regions” or CDRs. As used herein, “frameworkregion” or “FR” refers to a region in the variable domain which islocated between the CDRs. As used herein, “complementary determiningregion” or “CDR” refers to variable regions in VHHs that contains theamino acid sequences capable of specifically binding to antigenictargets.

In various embodiments, the targeting moiety directed against CD8comprises a VHH having a variable domain comprising at least one CDR1,CDR2, and/or CDR3 sequences.

In some embodiments, the CDR1 sequence is selected from:

(SEQ ID NO: 12) GFTFDDYAMS or (SEQ ID NO: 13) GFTFDDYAIG.

In some embodiments, the CDR2 sequence is selected from:

(SEQ ID NO: 14) TINWNGGSAEYAEPVKG or (SEQ ID NO: 15) CIRVSDGSTYYADPVKG.

In some embodiments, the CDR3 sequence is selected from:

(SEQ ID NO: 16) KDADLVWYNLS or (SEQ ID NO: 17) KDADLVWYNLR or (SEQ IDNO: 18) AGSLYTCVQSIVVVPARPYYDMDY.

In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:12,SEQ ID NO:14, and SEQ ID NO:16. In various embodiments, the CD8targeting moiety comprises SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:17.In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:12,SEQ ID NO:14, and SEQ ID NO:18.

In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:12,SEQ ID NO:15, and SEQ ID NO:16. In various embodiments, the CD8targeting moiety comprises SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:17.In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:12,SEQ ID NO:15, and SEQ ID NO:18.

In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:13,SEQ ID NO:14, and SEQ ID NO:16. In various embodiments, the CD8targeting moiety comprises SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:17.In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:13,SEQ ID NO:14, and SEQ ID NO:18.

In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:13,SEQ ID NO:15, and SEQ ID NO:16. In various embodiments, the CD8targeting moiety comprises SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17.In various embodiments, the CD8 targeting moiety comprises SEQ ID NO:13,SEQ ID NO:15, and SEQ ID NO:18.

In various embodiments, the CD8 targeting moiety comprises an amino acidsequence selected from the following sequences:

R3HCD27 (SEQ ID NO: 19) QVQLQESGGGSVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSTINWNGGSAEYAEPVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYCAKDADLVWYNLSTGQGTQVTVSSAAAYPYDVPDYGS or R3HCD129 (SEQ ID NO: 20)QVQLQESGGGLVQPGGSLRLSCAASGFTFDDYAMSWVRQVPGKGLEWVSTINWNGGSAEYAEPVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYCAKDADLVWYNLRTGQGTQVTVSSAAAYPYDVPDYGS or R2HCD26 (SEQ ID NO: 21)QVQLQESGGGLVQAGGSLRLSCAASGFTFDDYAIGWFRQAPGKEREGVSCIRVSDGSTYYADPVKGRFTISSDNAKNTVYLQMNSLKPEDAAVYYCAAGSLYTCVQSIVVVPARPYYDMDYWGKGTQVTVSSAAAYPYDVPDYGS.

In various embodiments, the targeting moiety comprises an amino acidsequence described in US Patent Publication No. 2014/0271462, the entirecontents of which are incorporated by reference. In various embodiments,the CD8 targeting moiety comprises an amino acid sequence described inTable 0.1, Table 0.2, Table 0.3, and/or FIGS. 1A-12I of US PatentPublication No. 2014/0271462, the entire contents of which areincorporated by reference. In various embodiments, the CD8 targetingmoiety comprises a HCDR1 of a HCDR1 of SEQ ID NO: 22 or 23 and/or aHCDR2 of HCDR1 of SEQ ID NO: 22 or 23 and/or a HCDR3 of HCDR1 of SEQ IDNO: 22 or 23 and/or a LCDR1 of LCDR1 of SEQ ID NO: 24 and/or a LCDR2 ofLCDR1 of SEQ ID NO: 24 and/or a LCDR3 of LCDR1 of SEQ ID NO: 24, asprovided below.

SEQ ID NO: 22: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp ThrTyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala ArgIle Asp Pro Ala Asn Asp Asn Thr Leu Tyr Ala Ser Lys Phe Gln Gly Arg AlaThr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Gly Arg Gly Tyr Gly Tyr Tyr ValPhe Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser. SEQ ID NO: 23:Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr ValLys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His TrpVal Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met Gly Arg Ile Asp Pro AlaAsn Asp Asn Thr Leu Tyr Ala Ser Lys Phe Gln Gly Arg Val Thr Ile Thr AlaAsp Thr Ser Thr Asp Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu AspThr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Gly Tyr Tyr Val Phe Asp His TrpGly Gln Gly Thr Leu Val Thr Val Ser Ser. SEQ ID NO: 24: Asp Val Gln IleThr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile ThrCys Arg Thr Ser Arg Ser Ile Ser Gln Tyr Leu Ala Trp Tyr Gln Gln Lys ProGly Lys Val Pro Lys Leu Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly ValPro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile SerSer Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln His Asn Glu AsnPro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys.

In various embodiments, the present invention contemplates the use ofany natural or synthetic analogs, mutants, variants, alleles, homologsand orthologs (herein collectively referred to as “analogs”) of thetargeting moiety directed against CD8 α s described herein. In variousembodiments, the amino acid sequence of the targeting moiety thattargets CD8 further includes an amino acid analog, an amino acidderivative, or other non-classical amino acids.

In various embodiments, the targeting moiety of the invention maycomprise an amino acid sequence that targets CD8 which is at least about60%, at least about 61%, at least about 62%, at least about 63%, atleast about 64%, at least about 65%, at least about 66%, at least about67%, at least about 68%, at least about 69%, at least about 70%, atleast about 71%, at least about 72%, at least about 73%, at least about74%, at least about 75%, at least about 76%, at least about 77%, atleast about 78%, at least about 79%, at least about 80%, at least about81%, at least about 82%, at least about 83%, at least about 84%, atleast about 85%, at least about 86%, at least about 87%, at least about88%, at least about 89%, at least about 90%, at least about 91%, atleast about 92%, at least about 93%, at least about 94%, at least about95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99%, or 100% identical with any one of the sequencesdisclosed herein (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, about 99% or about 100% sequence identity with any one of thesequences disclosed herein).

In various embodiments, the targeting moiety of the invention comprisesone or more mutations that do not substantially reduce the present CD8targeting moiety's capability to specifically bind to CD8. In variousembodiments, the mutations do not substantially reduce the targetingmoiety's capability to specifically bind to CD8 without functionallymodulating CD8.

In various embodiments, the binding affinity of the CD8 targeting moietyfor the full-length and/or mature forms and/or isoforms and/or splicevariants and/or fragments and/or any other naturally occurring orsynthetic analogs, variants, or mutants (including monomeric, dimeric,heterodimeric, multimeric and/or associated forms) of human CD8 α and/orβ chains may be described by the equilibrium dissociation constant(K_(D)). In various embodiments, the present chimeric protein comprisesa targeting moiety that binds to the full-length and/or mature formsand/or isoforms and/or splice variants and/or fragments and/or any othernaturally occurring or synthetic analogs, variants, or mutants(including monomeric, dimeric, heterodimeric, multimeric and/orassociated forms) of human CD8 α and/or β chains with a K_(D) of lessthan about 1 uM, about 900 nM, about 800 nM, about 700 nM, about 600 nM,about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM,about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or about 1nM.

In various embodiments, the present chimeric protein comprises atargeting moiety that binds but does not functionally modulate theantigen of interest, i.e., CD8. For instance, in various embodiments,the targeting moiety simply targets the antigen but does notsubstantially functionally modulate the antigen, e.g. it does notsubstantially inhibit, reduce or neutralize a biological effect that theantigen has. In various embodiments, the targeting moiety agent binds anepitope that is physically separate from an antigen site that isimportant for its biological activity (e.g. an antigen's active site).

Such non-functionally modulating (e.g. non-neutralizing) binding findsuse in various embodiments of the present invention, including methodsin which the present chimeric protein is used to directly or indirectlyrecruit active immune cells to a site of need via an effector antigen.For example, in various embodiments, the present chimeric protein may beused to directly or indirectly recruit cytotoxic T cells via CD8 to atumor cell in a method of reducing or eliminating a tumor (e.g. thechimeric protein may comprise a targeting moiety having an anti-CD8antigen recognition domain and a targeting moiety having a recognitiondomain (e.g. an antigen recognition domain) directed against a tumorantigen or receptor). In such embodiments, it is desirable to directlyor indirectly recruit CD8-expressing cytotoxic T cells but not toneutralize the CD8 activity. In these embodiments, CD8 signaling is animportant piece of the tumor reducing or eliminating effect.

By way of non-limiting example, in various embodiments, the presentchimeric protein has a targeting moiety directed against a checkpointmarker expressed on a T cell, e.g. one or more of PD-1, CD28, CTLA4,ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

For example, in some embodiments, the targeting moiety comprises anantigen recognition domain that specifically binds to a target (e.g.antigen, receptor) associated with B cells. In some embodiments, therecognition domains directly or indirectly recruit B cells, e.g., insome embodiments, to a therapeutic site (e.g. a locus with one or moredisease cell or cell to be modulated for a therapeutic effect).Illustrative B cell antigens of interest include, for example, CD10,CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72,CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83,CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, CDw150, andB-cell maturation antigen (BCMA). In various embodiments, a targetingmoiety of the chimeric protein binds one or more of these illustrative Bcell antigens.

By way of further example, in some embodiments, the targeting moietycomprises an antigen recognition domain that specifically binds to atarget (e.g. antigen, receptor) associated with Natural Killer cells. Insome embodiments, the recognition domains directly or indirectly recruitNatural Killer cells, e.g., in some embodiments, to a therapeutic site(e.g. a locus with one or more disease cell or cell to be modulated fora therapeutic effect). Illustrative Natural Killer cell antigens ofinterest include, for example TIGIT, 2B4/SLAMF4, KIR2DS4, CD155/PVR,KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7,LMIR3/CD300LF, Kir1alpha, DNAM-1, LMIR5/CD300LB, Fc-epsilon RII,LMIR6/CD300LE, Fc-γ RI/CD64, MICA, Fc-γ RIIB/CD32b, MICB, Fc-γRIIC/CD32c, MULT-1, Fc-γ RIIA/CD32a, Nectin-2/CD112, Fc-γ RIII/CD16,NKG2A, FcRH1/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30,FcRH5/IRTA2, NKp44, Fc-Receptor-like 3/CD16-2, NKp46/NCR1, NKp80/KLRF1,NTB-A/SLAMF6, Rae-1, Rae-1 α, Rae-1 β, Rae-1 delta, H60, Rae-1 epsilon,ILT2/CD85j, Rae-1 γ, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a,TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2,KIR2DL4/CD158d and ULBP-3. In various embodiments, a targeting moiety ofthe chimeric protein binds one or more of these illustrative NK cellantigens.

Also, in some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with macrophages/monocytes. In some embodiments,the recognition domains directly or indirectly recruitmacrophages/monocytes, e.g., in some embodiments, to a therapeutic site(e.g. a locus with one or more disease cell or cell to be modulated fora therapeutic effect). Illustrative macrophages/monocyte antigens ofinterest include, for example SIRP1a, B7-1/CD80, ILT4/CD85d, B7-H1,ILT5/CD85a, Common β Chain, Integrin α 4/CD49d, BLAME/SLAMF8, Integrin αX/CDIIc, CCL6/C10, Integrin β 2/CD18, CD155/PVR, Integrin β 3/CD61,CD31/PECAM-1, Latexin, CD36/SR-B3, Leukotriene B4 R1, CD40/TNFRSF5,LIMPIIISR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68,LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163,LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2,EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB, Fc-γ RI/CD64,Osteopontin, Fc-γ RIIB/CD32b, PD-L2, Fc-γ RIIC/CD32c, Siglec-3/CD33,Fc-γ RIIA/CD32a, SIGNR1/CD209, Fc-γ RIII/CD16, SLAM, GM-CSF R α,TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ RI, TLR4, IFN-gannna R2, TREM-I,IL-I RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1,2B4/SLAMF 4, IL-10 R α, ALCAM, IL-10 R β, AminopeptidaseN/ANPEP,ILT2/CD85j, Common β Chain, ILT3/CD85k, Clq R1/CD93, ILT4/CD85d, CCR1,ILT5/CD85a, CCR2, Integrin α 4/CD49d, CCR5, Integrin α M/CDII b, CCR8,Integrin α X/CDIIc, CD155/PVR, Integrin β 2/CD18, CD14, Integrin β3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, Leukotriene B4-R1, CD68,LIMPIIISR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163,LMIR3/CD300LF, Coagulation Factor III/Tissue Factor, LMIR5/CD300LB,CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R,DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105,NCAM-L1, Fc-γ RI/CD64, PSGL-1, Fc-γ RIIIICD16, RP105, G-CSF R,L-Selectin, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54,TCCR/WSX-1, ICAM-2/CD102, TREM-I, IL-6 R, TREM-2, CXCRI/IL-8 RA, TREM-3and TREMLI/TLT-1. In various embodiments, a targeting moiety of thechimeric protein binds one or more of these illustrativemacrophage/monocyte antigens.

Also, in some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with dendritic cells. In some embodiments, therecognition domains directly or indirectly recruit dendritic cells,e.g., in some embodiments, to a therapeutic site (e.g. a locus with oneor more disease cell or cell to be modulated for a therapeutic effect).Illustrative dendritic cell antigens of interest include, for example,CLEC9A, XCR1, RANK, CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163,SR-A1/MSR, CD5L, SREC-1, CL-PI/COLEC12, SREC-II, LIMPIIISRB2, RP105,TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB Ligand/TNFSF9,IL-12/IL-23 p40, 4-Amino-1,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine,ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, lutegrin α4/CD49d, Aag, Integrin β 2/CD18, AMICA, Langerin, B7-2/CD86, LeukotrieneB4 RI, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93,LMIR3/CD300LF, CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5,MAG/Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A,CD84/SLAMF5, MMR, CD97, NCAMLI, CD2F-10/SLAMF9, Osteoactivin GPNMB,Chern 23, PD-L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2/CD22,CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299,Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10,Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148,SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc-γ R1/CD64, TLR3, Fc-γRIIB/CD32b, TREM-1, Fc-γ RIIC/CD32c, TREM-2, Fc-γ RIIA/CD32a, TREM-3,Fc-γ RIII/CD16, TREML1/TLT-1, ICAM-2/CD102, DEC205, and Vanilloid R1. Invarious embodiments, a targeting moiety of the chimeric protein bindsone or more of these illustrative DC antigens.

In an exemplary embodiment, the present chimeric protein comprises atargeting moiety directed against Clec9A. In various embodiments, thetargeting moiety directed against Clec9A is a protein-based agentcapable of specific binding to Clec9A. In various embodiments, thetargeting moiety is a protein-based agent capable of specific binding toClec9A without neutralization of Clec9A. Clec9A is a group V C-typelectin-like receptor (CTLR) expressed on the surface of a subset ofdendritic cells (i.e., BDCA₃+ dendritic cells) specialized for theuptake and processing of materials from dead cells. Clec9A recognizes aconserved component within nucleated and nonnucleated cells, exposedwhen cell membranes are damaged. CLEC9A is expressed at the cell surfaceas a glycosylated dimer and can mediate endocytosis, but notphagocytosis. CLEC9A possesses a cytoplasmic immunoreceptortyrosine-based activation-like motif that can recruit Syk kinase andinduce proinflammatory cytokine production (see Huysamen et al. (2008),JBC, 283:16693-701).

In various embodiments, the present chimeric protein comprises atargeting moiety having an antigen recognition domain that recognizes anepitope present on Clec9A. In an embodiment, the antigen-recognitiondomain recognizes one or more linear epitopes present on Clec9A. As usedherein, a linear epitope refers to any continuous sequence of aminoacids present on Clec9A. In another embodiment, the antigen-recognitiondomain recognizes one or more conformational epitopes present on Clec9A.As used herein, a conformation epitope refers to one or more sections ofamino acids (which may be discontinuous) which form a three-dimensionalsurface with features and/or shapes and/or tertiary structures capableof being recognized by an antigen recognition domain.

In various embodiments, the present chimeric protein comprises atargeting moiety that may bind to the full-length and/or mature formsand/or isoforms and/or splice variants and/or fragments and/or any othernaturally occurring or synthetic analogs, variants, or mutants of humanClec9A. In various embodiments, the targeting moiety may bind to anyforms of the human Clec9A, including monomeric, dimeric, heterodimeric,multimeric and associated forms. In an embodiment, the targeting moietybinds to the monomeric form of Clec9A. In another embodiment, thetargeting moiety binds to a dimeric form of Clec9A. In a furtherembodiment, the targeting moiety binds to glycosylated form of Clec9A,which may be either monomeric or dimeric.

In an embodiment, the present chimeric protein comprises a targetingmoiety with an antigen recognition domain that recognizes one or moreepitopes present on human Clec9A. In an embodiment, the human Clec9Acomprises the amino acid sequence of:

(SEQ ID NO: 25) MHEEEIYTSLQWDSPAPDTYQKCLSSNKCSGACCLVMVISCVFCMGLLTASIFLGVKLLQVSTIAMQQQEKLIQQERALLNFTEWKRSCALQMKYCQAFMQNSLSSAHNSSPCPNNWIQNRESCYYVSEIWSIWHTSQENCLKEGSTLLQIESKEEMDFITGSLRKIKGSYDYWVGLSQDGHSGRWLWQDGSSPSPGLLPAERSQSANQVCGYVKSNSLLSSNCSTWKYFICEKYALRSSV.

In various embodiments, the present chimeric protein comprises atargeting moiety capable of specific binding. In various embodiments,the chimeric protein comprises a targeting moiety having an antigenrecognition domain such as an antibody or derivatives thereof. In anembodiment, the chimeric protein comprises a targeting moiety which isan antibody. In various embodiments, the antibody is a full-lengthmultimeric protein that includes two heavy chains and two light chainsas described elsewhere herein. In some embodiments, the antibody is achimeric antibody. In some embodiments, the antibody is a humanizedantibody.

In some embodiments, the present chimeric protein comprises a targetingmoiety which is a single-domain antibody, such as a VHH (which iscommercially available under the trademark of NANOBODIES) as describedelsewhere herein. In an embodiment, the present chimeric proteincomprises a VHH.

In some embodiments, the present chimeric protein comprises a targetingmoiety which is a VHH comprising a single amino acid chain having four“framework regions” or FRs and three “complementary determining regions”or CDRs. As used herein, “framework region” or “FR” refers to a regionin the variable domain which is located between the CDRs. As usedherein, “complementary determining region” or “CDR” refers to variableregions in VHHs that contains the amino acid sequences capable ofspecifically binding to antigenic targets.

In various embodiments, the present chimeric protein comprises a VHHhaving a variable domain comprising at least one CDR1, CDR2, and/or CDR3sequences.

In some embodiments, the CDR1 sequence is selected from:

(SEQ ID NO: 26) GSISSINVMG; (SEQ ID NO: 27) GSFSSINVMG; (SEQ ID NO: 28)GSISSINIMG; (SEQ ID NO: 29) GSISSINIMG; (SEQ ID NO: 30) VSIFSINAMG; (SEQID NO: 31) GSIFSLNAMG; (SEQ ID NO: 32) GRTISNYDMA; (SEQ ID NO: 33)GRTFTTSLMQ; (SEQ ID NO: 34) ERNLRIYDMA; (SEQ ID NO: 35) ERNLRSYDMA; (SEQID NO: 36) GLTFSNYHMG; (SEQ ID NO: 37) GLTFSSYHMG; (SEQ ID NO: 38)GLTFSRYHMG; (SEQ ID NO: 39) GLTLSSYYIA; (SEQ ID NO: 40) GLTFSSYYTG; (SEQID NO: 41) GLTLSSYHMG; (SEQ ID NO: 42) GRTSSPYVTG; (SEQ ID NO: 43)GFTFSGYVMS; (SEQ ID NO: 44) GFTFSGYVMT; or (SEQ ID NO: 45) GFTFSGYLMS.

In some embodiments, the CDR2 sequence is selected from:

(SEQ ID NO: 46) RITNLGLPNYADWLKD; (SEQ ID NO: 47) RITNLGLPNYADSVTG; (SEQID NO: 48) RITNIGLPNYADSVKG; (SEQ ID NO: 49) RITNLGLPNYADSVEG; (SEQ IDNO: 50) AITSGGRVVYSDSVKG; (SEQ ID NO: 51) AITSGGRTAYADSVKG; (SEQ ID NO:52) HITSDGRIVYADPVKG; (SEQ ID NO: 53) RISGSGDRTDYADSVKG; (SEQ ID NO: 54)SITWSTGNTHYADSVKG; (SEQ ID NO: 55) VISSSGDSTHYSDFVKG; (SEQ ID NO: 56)VITSSGDSTHYSDFVKG; (SEQ ID NO: 57) QITWSDASIYYAGSVKG; (SEQ ID NO: 58)QITWSDTSIYYAGSVKG; (SEQ ID NO: 59) QITWSDGTTYYPGSVKG; (SEQ ID NO: 60)QIRWSDDSTYYPGSVKG; (SEQ ID NO: 61) QISWSDDSTYYADSVKG; (SEQ ID NO: 62)TVSWGGVTYYADSVKG; (SEQ ID NO: 63) SIGSGGGYPSYTDSVEG; (SEQ ID NO: 64)SIGSGGGYPSYTGSVEG; (SEQ ID NO: 65) HIGSGGGYPSYTDSVQG; (SEQ ID NO: 66)HIGSGGGHATYTDSVEG; or (SEQ ID NO: 67) TIGSGGGITSYADSVKG.

In some embodiments, the CDR3 sequence is selected from:

(SEQ ID NO: 68) VALSAEY; (SEQ ID NO: 69) VALKAEY; (SEQ ID NO: 70)VGLKAEY; (SEQ ID NO: 71) KTKSAVLFGGMDY; (SEQ ID NO: 72) YIRGEDY; (SEQ IDNO: 73) KHYASNY; (SEQ ID NO: 74) QDFGSPSF; (SEQ ID NO: 75) QDFRSPDF;(SEQ ID NO: 76) QIFGSPNF; (SEQ ID NO: 77) LAIHGDY; (SEQ ID NO: 78)NQIRQWP; (SEQ ID NO: 79) NSIRQWP; (SEQ ID NO: 80) NAIRQWP; (SEQ ID NO:81) RKVGGPDY; (SEQ ID NO: 82) NTFGNVY; LGR; or VIK.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:26,SEQ ID NO:46, and SEQ ID NO:68.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:27,SEQ ID NO:47, and SEQ ID NO:69.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:28,SEQ ID NO:48, and SEQ ID NO:69.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:26,SEQ ID NO:49, and SEQ ID NO:70.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:29,SEQ ID NO:50, and SEQ ID NO:71.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:30,SEQ ID NO:51, and SEQ ID NO:72.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:31,SEQ ID NO:52, and SEQ ID NO:73.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:32,SEQ ID NO:53, and SEQ ID NO:74.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:32,SEQ ID NO:53, and SEQ ID NO:75.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:32,SEQ ID NO:53, and SEQ ID NO:76.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:33,SEQ ID NO:54, and SEQ ID NO:77.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:34,SEQ ID NO:55, and SEQ ID NO:78.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:34,SEQ ID NO:55, and SEQ ID NO:79.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:35,SEQ ID NO:56, and SEQ ID NO:80.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:36,SEQ ID NO:57, and SEQ ID NO:81.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:37,SEQ ID NO:58, and SEQ ID NO:81.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:38,SEQ ID NO:59, and SEQ ID NO:81.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:39,SEQ ID NO:60, and SEQ ID NO:81.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:40,SEQ ID NO:61, and SEQ ID NO:81.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:41,SEQ ID NO:61, and SEQ ID NO:81.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:42,SEQ ID NO:62, and SEQ ID NO:82.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:43,SEQ ID NO:63, and LGR.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:43,SEQ ID NO:64, and LGR.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:43,SEQ ID NO:65, and LGR.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:44,SEQ ID NO:66, and LGR.

In some embodiments, the Clec9A targeting moiety comprises SEQ ID NO:45,SEQ ID NO:67, and VlK.

In various embodiments, the Clec9A targeting moiety comprises an aminoacid sequence selected from the following sequences:

R2CHCL8 (SEQ ID NO: 83)QVQLVESGGGLVHPGGSLRLSCAASGSISSINVMGWYRQAPGKERELVARITNLGLPNYADWLKDRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYLVAL SAEYWGQGTQVTVSS;R1CHCL50 (SEQ ID NO: 84)QVQLVESGGGLVHPGGSLRLSCAASGSFSSINVMGWYRQAPGKERELVARITNLGLPNYADSVTGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCYLVAL KAEYWGQGTQVTVSS;R1CHCL21 (SEQ ID NO: 85)QVQLVESGGGLVHRGGSLRLSCAASGSISSINIMGWYRQAPGKERELVARITNIGLPNYADSVKGRFTISRDNAKSTVYLQMNSLNAEDTAVYYCYLVAL KAEYWGQGTQVTVSS;R2CHCL87 (SEQ ID NO: 86)QVQLVESGGGLVQPGGSLRLSCAASGSISSINVMGWYRQAPGKERELVARITNLGLPNYADSVEGRFTISRDKDENTVYLEMNTLKPEDTAVYYCYLVGL KAEYWGQGTQVTVSS;R2CHCL24 (SEQ ID NO: 87)QVQLVESGGGLVQPGGSLRLSCAASGSSDSINAMGWYRQAPGKERELVAAITSGGRVVYSDSVKGRGTISRDNAKNTVYLQIASLKPEDTAVYYCNVKTKSAVLFGGMDYWGKGTQVTVSS; R2CHCL38 (SEQ ID NO: 88)QVQLVESGGGLVQPGGSLRLSCAASVSIFSINAMGWYRQAPGKERELVAAITSGGRTAYADSVKGRFTISRDNSKNTVYLQMDSLKPEDTDVYYCKAYIR GEDYWGKGTQVTVSS;R1CHCL16 (SEQ ID NO: 89)DVQLVESGGGLVQPGGSLRLSCAASGSIFSLNAMGWYRQAPGKERELVAHITSDGRIVYADPVKGRFTISRVDGKNMVTLQMNSLKPEDTAVYYCNAKHY ASNYWGQGTQVTVSS;R2CHCL10 (SEQ ID NO: 90)QVQLVESGGGSVQAGGSLRLSCAASGRTISNYDMAWSRQAPGKEREFVARISGSGDRTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCQIQD FGSPSFSGQGTQVTVSS;R1CHCL34 (SEQ ID NO: 91)DVQLVESGGGSVQAGGSLRLSCAASGRTISNYDMAWSRQAPGKEREFVARISGSGDRTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCQIQD FRSPDFWSQGTQVTVSS;R1CHCL82 (SEQ ID NO: 92)QVQLVESGGESVQAGGSLRLSCAASGRTISNYDMAWSRQAPGKEREFVARISGSGDRTDYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYNCQTQI FGSPNFSGQGTQVTVSS;R2CHCL3 (SEQ ID NO: 93)QVQLVESGGGLVQAGDSLRLSCAASGRTFTTSLMQWHRQAPGKEREFVASITWSTGNTHYADSVKGRFTISRDNARNTVYLQMNSLKPEDTAIYTCRVLA IHGDYWGQGTQVTVSS;R2CHCL69 (SEQ ID NO: 94)DVQLVESGGGLVQAGDSLRLSCAASERNLRIYDMAWYRQAPGKEREYVAVISSSGDSTHYSDFVKGRFTISRDNAKNTVSLQMDSLKPEDTAFYYCNVNQ IRQWPWGQGTQVTVSS;R1CHCL56 (SEQ ID NO: 95)QVQLVESGGGLVQAGDSLRLSCAASERNLRIYDMAWYRQAPGKEREYVAVISSSGDSTHYSDFVKGRFTISRDNAKNTVSLQMDSLKPEDTAFYYCNVNS IRQWPWGQGTQVTVSS;R2CHCL32 (SEQ ID NO: 96)QVQLVESGGGLVQAGDSLRLSCTASERNLRSYDMAWWRQAPGKEREYVAVITSSGDSTHYSDFVKGRFTISRDNAKNTVSLQMDSLKPEDTASYYCNVNA IRQWPWGQGTQVTVSS;R2CHCL49 (SEQ ID NO: 97)DVQLVESGGGSVQAGGSLRLSCAISGLTFSNYHMGWYRQAPGREREFVAQITWSDASIYYAGSVKGRFTISRDNVKNIVYLQIDNLKPEDTAIYYCDARK VGGPDYWGQGTQVTVSS;R2CHCL53 (SEQ ID NO: 98)QVQLVESGGGLVQAGGSLTLSCAISGLTFSSYHMGWYRQAPGREREFVAQITWSDTSIYYAGSVKGRFTISRDNVKNIVYLQIDNLKPEDTAIYYCDARK VGGPDYWGQGTQVTVSS;R2CHCL22 (SEQ ID NO: 99)DVQLVESGGGLVQAGGSLRLSCAISGLTFSRYHMGWYRQAPGREREFVAQITWSDGTTYYPGSVKGRFTISRDNARNTVYLQIDNLKPEDTAIYYCDARK VGGPDYWGQGTQVTVSS;R2CHCL25 (SEQ ID NO: 100)QVQLVESGGGLVQAGGSLRLSCATSGLTLSSYYIAWYRQAPGREREFVAQIRWSDDSTYYPGSVKGRFTISRDNARNTVYLRMDNLKPEDTARYYCDARK VGGPDYWGQGTQVTVSS;R2CHCL18 (SEQ ID NO: 101)DVQLVESGGGLVQAGGSLRLSCATSGLTFSSYYTGWYRQAPGREREFVAQISWSDDSTYYADSVKGRFTISRDNARNTVYLQMNNLKPGDTAIYYCDARK VGGPDYWGQGTQVTVSS;R1CHCL23 (SEQ ID NO: 102)DVQLVESGGGLVQAGGSLRLSCATSGLTLSSYHMGWYRQAPGREREFVAQISWSDDSTYYADSVKGRFTISRDNARNTVYLQMNNLKPEDTAIYYCDARK VGGPDYWGQGTQVTVSS;R1CHCL27 (SEQ ID NO: 103)DVQLVESGGGLVQAGGSLRLSCAASGRTSSPYVTGWYRQTPGKEREPVATVSWGGVTYYADSVKGRFTISRDNAKNTVYLQMNALKPEDTAIYYCNVNTF GNVYWGQGTQVTVSS;R2CHCL13 (SEQ ID NO: 104)QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYVMSWVRQAPGKGLEWVASIGSGGGYPSYTDSVEGRFTISRDNAKNTLYLLMDNLKPDDTAVYYCEMLG RRGQGTQVTVSS;R2CHCL14 (SEQ ID NO: 105)QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYVMSWVRQAPGKGLEWVASIGSGGGYPSYTDSVEGRFTISRDNAKNTLYLQMNNLKPDDTAVYYCEMLG RRGQGTQVTVSS;R2CHCL42 (SEQ ID NO: 106)QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYVMSWVRQAPGKGLEWVASIGSGGGYPSYTGSVEGRFTISRDNAKNTLYLLMNNLKPDDTAVYYCEMLG RRGQGTQVTVSS;R2CHCL41 (SEQ ID NO: 107)QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYVMSWVRQAPGKGLEWVAHIGSGGGYPSYTDSVQGRFTISRDNAKNTLYLQMNNLKPEDTAVYYCEMLG RRGQGTQVTVSS;R2CHCL94 (SEQ ID NO: 108)QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYVMTWVRQAPGKGLEWVAHIGSGGGHATYTDSVEGRFTISRDNAKNTLYLQMNNLKAEDTAVYYCEFLG RRGQGTQVTVSS; orR2CHCL27 (SEQ ID NO: 109)QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYLMSWVRQAPGKGLEWVATIGSGGGITSYADSVKGRFTISRDNAKNTLYLQMNNLKHEDTAVYYCETVI KRGQGTQVTVSS.

In various embodiments, the present invention contemplates the use ofany natural or synthetic analogs, mutants, variants, alleles, homologsand orthologs (herein collectively referred to as “analogs”) of thetargeting moiety directed against Clec9A as described herein. In variousembodiments, the amino acid sequence of the targeting moiety directedagainst Clec9A further includes an amino acid analog, an amino acidderivative, or other non-classical amino acids.

In various embodiments, the present chimeric protein comprises atargeting moiety comprising an amino acid sequence that is at least 60%identical to any one of the sequences disclosed herein. For example, thechimeric protein may comprise a targeting moiety comprising an aminoacid sequence that is at least about 60%, at least about 61%, at leastabout 62%, at least about 63%, at least about 64%, at least about 65%,at least about 66%, at least about 67%, at least about 68%, at leastabout 69%, at least about 70%, at least about 71%, at least about 72%,at least about 73%, at least about 74%, at least about 75%, at leastabout 76%, at least about 77%, at least about 78%, at least about 79%,at least about 80%, at least about 81%, at least about 82%, at leastabout 83%, at least about 84%, at least about 85%, at least about 86%,at least about 87%, at least about 88%, at least about 89%, at leastabout 90%, at least about 91%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98%, at least about 99%, or 100% identical toany one of the sequences discloses herein (e.g. about 60%, or about 61%,or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, orabout 67%, or about 68%, or about 69%, or about 70%, or about 71%, orabout 72%, or about 73%, or about 74%, or about 75%, or about 76%, orabout 77%, or about 78%, or about 79%, or about 80%, or about 81%, orabout 82%, or about 83%, or about 84%, or about 85%, or about 86%, orabout 87%, or about 88%, or about 89%, or about 90%, or about 91%, orabout 92%, or about 93%, or about 94%, or about 95%, or about 96%, orabout 97%, or about 98%, about 99% or about 100% sequence identity toany one of the sequences disclosed herein).

In various embodiments, the targeting moiety of the invention comprisesone or more mutations that do not substantially reduce the Clec9Atargeting moiety's capability to specifically bind to Clec9A. In variousembodiments, the mutations do not substantially reduce the targetingmoiety's capability to specifically bind to Clec9A without neutralizingClec9A.

In various embodiments, the binding affinity of the Clec9A targetingmoiety for the full-length and/or mature forms and/or isoforms and/orsplice variants and/or fragments and/or monomeric and/or dimeric formsand/or any other naturally occurring or synthetic analogs, variants, ormutants (including monomeric and/or dimeric forms) of human Clec9A maybe described by the equilibrium dissociation constant (K_(D)). Invarious embodiments, the present chimeric protein comprises a targetingmoiety that binds to the full-length and/or mature forms and/or isoformsand/or splice variants and/or fragments and/or any other naturallyoccurring or synthetic analogs, variants, or mutants (includingmonomeric and/or dimeric forms) of human Clec9A with a K_(D) of lessthan about 1 uM, about 900 nM, about 800 nM, about 700 nM, about 600 nM,about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM,about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about40 nM, about 30 nM, about 20 nM, about 10 nM, or about 5 nM, or about 1nM.

In various embodiments, the present chimeric protein comprises atargeting moiety that binds but does not functionally modulate theantigen of interest, e.g., Clec9A. For instance, in various embodiments,the Clec9A targeting moiety simply targets the antigen but does notsubstantially functionally modulate (e.g. substantially inhibit, reduceor neutralize) a biological effect that the antigen has. In variousembodiments, the Clec9A targeting moiety binds an epitope that isphysically separate from an antigen site that is important for itsbiological activity (e.g. an antigen's active site).

Such binding without significant function modulation finds use invarious embodiments of the present invention, including methods in whichthe present chimeric protein is used to directly or indirectly recruitactive immune cells to a site of need via an effector antigen. Forexample, in various embodiments, the present chimeric protein may beused to directly or indirectly recruit dendritic cells via Clec9A to atumor cell in a method of reducing or eliminating a tumor (e.g. thechimeric protein may comprise a targeting moiety having an anti-Clec9Aantigen recognition domain and a targeting moiety having a recognitiondomain (e.g. antigen recognition domain) directed against a tumorantigen or receptor). In such embodiments, it is desirable to directlyor indirectly recruit dendritic cells but not to functionally modulatethe Clec9A activity. In these embodiments, Clec9A signaling is animportant piece of the tumor reducing or eliminating effect.

In some embodiments, the present chimeric protein enhancesantigen-presentation by dendritic cells. For example, in variousembodiments, the present chimeric protein may directly or indirectlyrecruits dendritic cells via Clec9A to a tumor cell, where tumorantigens are subsequently endocytosed and presented on the dendriticcell for induction of potent humoral and cytotoxic T cell responses.

In some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) on immune cells selected from, but not limited to,megakaryocytes, thrombocytes, erythrocytes, mast cells, basophils,neutrophils, eosinophils, or subsets thereof. In some embodiments, therecognition domains directly or indirectly recruit megakaryocytes,thrombocytes, erythrocytes, mast cells, basophils, neutrophils,eosinophils, or subsets thereof, e.g., in some embodiments, to atherapeutic site (e.g. a locus with one or more disease cell or cell tobe modulated for a therapeutic effect).

In some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with megakaryocytes and/or thrombocytes.Illustrative megakaryocyte and/or thrombocyte antigens of interestinclude, for example, GP IIb/IIIa, GPIb, vWF, PF4, and TSP. In variousembodiments, a targeting moiety of the chimeric protein binds one ormore of these illustrative megakaryocyte and/or thrombocyte antigens.

In some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with erythrocytes. Illustrative erythrocyteantigens of interest include, for example, CD34, CD36, CD38, CD41a(platelet glycoprotein IIb/IIIa), CD41b (GPIIb), CD71 (transferrinreceptor), CD105, glycophorin A, glycophorin C, c-kit, HLA-DR, H2(MHC-11), and Rhesus antigens. In various embodiments, a targetingmoiety of the chimeric protein binds one or more of these illustrativeerythrocyte antigens.

In some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with mast cells. Illustrative mast cells antigensof interest include, for example, SCFR/CD117, F_(cε)RI, CD2, CD25, CD35,CD88, CD203c, C5R1, CMAI, FCERIA, FCER2, TPSABI. In various embodiments,a targeting moiety of the chimeric protein binds one or more of thesemast cell antigens.

In some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with basophils. Illustrative basophils antigens ofinterest include, for example, F_(cε)RI, CD203c, CD123, CD13, CD107a,CD107b, and CD164. In various embodiments, a targeting moiety of thechimeric protein binds one or more of these basophil antigens.

In some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with neutrophils. Illustrative neutrophils antigensof interest include, for example, 7D5, CD10/CALLA, CD13, CD16 (FcRIII),CD18 proteins (LFA-1, CR3, and p150, 95), CD45, CD67, and CD177. Invarious embodiments, a targeting moiety of the chimeric protein bindsone or more of these neutrophil antigens.

In some embodiments, the targeting moiety comprises an antigenrecognition domain that specifically binds to a target (e.g. antigen,receptor) associated with eosinophils. Illustrative eosinophils antigensof interest include, for example, CD35, CD44 and CD69. In variousembodiments, a targeting moiety of the chimeric protein binds one ormore of these eosinophil antigens.

In various embodiments, the targeting moiety may comprise an antigenrecognition domain may bind to any appropriate target, antigen,receptor, or cell surface markers known by the skilled artisan. In someembodiments, the antigen or cell surface marker is a tissue-specificmarker. Illustrative tissue-specific markers include, but are notlimited to, endothelial cell surface markers such as ACE, CD14, CD34,CDH5, ENG, ICAM2, MCAM, NOS3, PECAMI, PROCR, SELE, SELP, TEK, THBD,VCAMI, VWF; smooth muscle cell surface markers such as ACTA2, MYHIO,MYHI 1, MYH9, MYOCD; fibroblast (stromal) cell surface markers such asALCAM, CD34, COLIAI, COL1A2, COL3A1, FAP, PH-4; epithelial cell surfacemarkers such as CDID, K6IRS2, KRTIO, KRT13, KRT17, KRT18, KRT19, KRT4,KRT5, KRT8, MUCI, TACSTDI; neovasculature markers such as CD13, TFNA,Alpha-v beta-3 (α_(v)β₃), E-selectin; and adipocyte surface markers suchas ADIPOQ, FABP4, and RETN. In various embodiments, a targeting moietyof the chimeric protein binds one or more of these antigens. In variousembodiments, a targeting moiety of the chimeric protein binds one ormore of cells having these antigens.

In some embodiments, the recognition domains specifically bind to atarget (e.g. antigen, receptor) associated with tumor cells. In someembodiments, the recognition domains directly or indirectly recruittumor cells. For instance, in some embodiments, the direct or indirectrecruitment of the tumor cell is to one or more effector cell (e.g. animmune cell as described herein) that can kill and/or suppress the tumorcell.

Tumor cells, or cancer cells refer to an uncontrolled growth of cells ortissues and/or an abnormal increased in cell survival and/or inhibitionof apoptosis which interferes with the normal functioning of bodilyorgans and systems. For example, tumor cells include benign andmalignant cancers, polyps, hyperplasia, as well as dormant tumors ormicrometastases. Illustrative tumor cells include, but are not limitedto cells of: basal cell carcinoma, biliary tract cancer; bladder cancer;bone cancer; brain and central nervous system cancer; breast cancer;cancer of the peritoneum; cervical cancer; choriocarcinoma; colon andrectum cancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney or renal cancer; larynx cancer; leukemia; liver cancer; lungcancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as wellas B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (e.g. that associated with brain tumors), andMeigs' syndrome.

Tumor cells, or cancer cells also include, but are not limited to,carcinomas, e.g. various subtypes, including, for example,adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, andtransitional cell carcinoma), sarcomas (including, for example, bone andsoft tissue), leukemias (including, for example, acute myeloid, acutelymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell),lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkinlymphomas, light chain, non-secretory, MGUS, and plasmacytomas), andcentral nervous system cancers (including, for example, brain (e.g.gliomas (e.g. astrocytoma, oligodendroglioma, and ependymoma),meningioma, pituitary adenoma, and neuromas, and spinal cord tumors(e.g. meningiomas and neurofibroma).

Illustrative tumor antigens include, but are not limited to,MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosinedeaminase-binding protein (ADAbp), cyclophilin b, Colorectal associatedantigen (CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA) and itsimmunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate SpecificAntigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3,prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zetachain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3,MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family oftumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6,GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4,tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein,E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117,PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC),fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides,viral products such as human papilloma virus proteins, Smad family oftumor antigens, Imp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brainglycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5,SCP-1 CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37, CD56, CD70,CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA, and BCMA(TNFRSF17). In various embodiments, a targeting moiety of the chimericprotein binds one or more of these tumor antigens.

In some embodiments, the present chimeric protein has (i) one or more ofthe targeting moieties which is directed against an immune cell selectedfrom a T cell, a B cell, a dendritic cell, a macrophage, a NK cell, orsubsets thereof and (ii) one or more of the targeting moieties which isdirected against a tumor cell, along with any of the modified (e.g.mutant) signaling agents described herein. In one embodiment, thepresent chimeric protein has (i) a targeting moiety directed against a Tcell (including, without limitation an effector T cell) and (ii) atargeting moiety is directed against a tumor cell, along with any of themodified (e.g. mutant) signaling agents described herein. In oneembodiment, the present chimeric protein has (i) a targeting moietydirected against a B cell and (ii) a targeting moiety is directedagainst a tumor cell, along with any of the modified (e.g. mutant)signaling agents described herein. In one embodiment, the presentchimeric protein has (i) a targeting moiety directed against a dendriticcell and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents describedherein. In one embodiment, the present chimeric protein has (i) atargeting moiety directed against a macrophage and (ii) a targetingmoiety is directed against a tumor cell, along with any of the modified(e.g. mutant) signaling agents described herein. In one embodiment, thepresent chimeric protein has (i) a targeting moiety directed against aNK cell and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a T cell,for example, mediated by targeting to CD8, SLAMF4, IL-2 R α,4-1BB/TNFRSF9, IL-2 R β, ALCAM, B7-1, IL-4 R, B7-H3, BLAME/SLAMFS,CEACAM1, IL-6 R, CCR3, IL-7 Rα, CCR4, CXCRI/IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-I 0 R β, CCRS, IL-12 R β 1, CCR9, IL-12 R β 2, CD2, IL-13 Rα 1, IL-13, CD3, CD4, ILT2/CDS5j, ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a,lutegrin α 4/CD49d, CDS, Integrin α E/CD103, CD6, Integrin α M/CD 11 b,CDS, Integrin α X/CD11c, Integrin β 2/CDIS, KIR/CD15S, CD27/TNFRSF7,KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31/PECAM-1,KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3,Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C,CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common γChain/IL-2 R γ, Osteopontin, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4,RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP β 1, CXCR4,SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1,EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, FcγRIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1B, TRAILRI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102,TRAILR3/TNFRSF10C, IFN-γR1, TRAILR4/TNFRSF10D, IFN-γ R2, TSLP, IL-1 R1,or TSLP R; and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein.

By way of non-limiting example, in various embodiments, the presentchimeric protein has a targeting moiety directed against (i) acheckpoint marker expressed on a T cell, e.g. one or more of PD-1, CD28,CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aRand (ii) a targeting moiety is directed against a tumor cell, along withany of the modified (e.g. mutant) signaling agents described herein.

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against PD-1.

In some embodiments, the chimeric protein has one or more targetingmoieties which selectively bind a PD-1 polypeptide. In some embodiments,the chimeric protein comprises one or more antibodies, antibodyderivatives or formats, peptides or polypeptides, or fusion proteinsthat selectively bind a PD-1 polypeptide.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibodypembrolizumab (aka MK-3475, KEYTRUDA), or fragments thereof.Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed inHamid, et al. (2013) New England Journal of Medicine 369 (2): 134-44,U.S. Pat. No. 8,354,509, and WO 2009/114335, the entire disclosures ofwhich are hereby incorporated by reference. In illustrative embodiments,pembrolizumab or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising the aminoacid sequence of:

(SEQ ID NO: 110) QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 111) EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody,nivolumab (aka BMS-936558, MDX-1106, ONO-4538, OPDIVO), or fragmentsthereof. Nivolumab (clone 5C4) and other human monoclonal antibodiesthat specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449and WO 2006/121168, the entire disclosures of which are herebyincorporated by reference. In illustrative embodiments, nivolumab or anantigen-binding fragment thereof comprises a heavy chain comprising theamino acid sequence of:

(SEQ ID NO: 112) QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAVIWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVTVSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGGPSVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFNSTYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEEMTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRWQEGNVFSCSV MHEALHNHYT QKSLSLSLGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 113) EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYDASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQ GTKVEIKRTVAAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibodypidilizumab (aka CT-011, hBAT or hBAT-1), or fragments thereof.Pidilizumab and other humanized anti-PD-I monoclonal antibodies aredisclosed in US 2008/0025980 and WO 2009/101611, the entire disclosuresof which are hereby incorporated by reference. In illustrativeembodiments, the anti-PD-1 antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a light chainvariable regions comprising an amino acid sequence selected from SEQ IDNOS: 15-18 of US 2008/0025980:

SEQ ID No: 15 of US 2008/0025980 (SEQ ID NO: 114):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWYQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK; SEQ ID No: 16of US 2008/0025980 (SEQ ID NO: 115):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTDYTLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK; SEQ ID No: 17of US 2008/0025980 (SEQ ID NO: 116):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTDYCLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK; SEQ ID No: 18of US 2008/0025980 (SEQ ID NO: 117):EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGG TKLEIK;

and/or a heavy chain comprising an amino acid sequence selected from SEQID NOS: 20-24 of US 2008/0025980:

SEQ ID No: 20 of US 2008/0025980 (SEQ ID NO: 118):QVQLVQSGSELKKPGASVKISCKASGYSFSNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVSTAYLQITSLTAEDTGMYFCAKVG YDALDYWGQGTLVTVSS;SEQ ID No: 21 of US 2008/0025980 (SEQ ID NO: 119):QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVSTAYLQITSLTAEDTGMYFCAKVG YDALDYWGQGTLVTVSS;SEQ ID No: 22 of US 2008/0025980 (SEQ ID NO: 120):QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVG YDALDYWGQGTLVTVSS;SEQ ID No: 23 of US 2008/0025980 (SEQ ID NO: 121):QIQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVG YDALDYWGQGTLVTVSS;SEQ ID No: 24 of US 2008/0025980 (SEQ ID NO: 122):QIQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTDSGESTYAEEFKGRFAFSLDTSVNTAYLQITSLNAEDTGMYFCVRVG YDALDYWGQGTLVTVSS.

In an embodiment, the targeting moiety comprises a light chaincomprising SEQ ID NO:18 of US 2008/0025980 and a heavy chain comprisingSEQ ID NO:22 of US 2008/0025980.

In an embodiment, the targeting moiety comprises AMP-514 (akaMEDI-0680).

In an embodiment, the targeting moiety comprises the PD-L2-Fc fusionprotein AMP-224, which is disclosed in WO2010/027827 and WO 2011/066342,the entire disclosures of which are hereby incorporated by reference. Insuch an embodiment, the targeting moiety may include a targeting domainwhich comprises SEQ ID NO:4 of WO2010/027827 (SEQ ID NO: 123):

LFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIALRKQLCQKLYSSKDTTKRPVTTTKREV NSAI

and/or the B7-DC fusion protein which comprises SEQ ID NO:83 ofWO2010/027827 (SEQ ID NO: 124):

MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQKVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVKASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVLRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK.

In an embodiment, the targeting moiety comprises the peptide AUNP 12 orany of the other peptides disclosed in US 2011/0318373 or 8,907,053. Forexample, the targeting moiety may comprise AUNP 12 (i.e., Compound 8 orSEQ ID NO:49 of US 2011/0318373) which has the sequence of SEQ ID NO:125:

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1E3, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E3 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 126) EVQLQQSGPV LVKPGASVKM SCKASGYTFT DYYMNWVKQS HGKSLEWIGNINPYNGGTTY NQKFKGKATL TVDKSSRTAY MEINSLTSED SAVYYCARGR IYDGSLDYWGQGTALTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 127) DIQMTQFPSS LCASQGGKVT VTCKASQDIN NYMAWYQHKP GKGPRLLIHYTSTLLSGIPS RFSGSGSGRD YSFSISNLEP EDIATYYCLQ YDNLWTFGGG TKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1E8, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E8 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 128) QVQLQQSGAE LAKPGASVRL SCKASGYTFT NYWMHWVKQR PGQGLEWIGHINPSSGFTTY NQNFKDKATL TADKSSNTAY MQLSSLTYED SAVYFCARED YDVDYWGQGTTLTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 129) DIVMTQSQKF MSTSVGDRVS VTCKASQSVD TNVAWYQQKP GQSPKALIFSASYRYSGVPD RFTGSGSGTD FTLTINSVQS EDLAEYFCQQ YNSYPYTFGS GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-1 antibody1H3, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1H3 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 130 EVQLVESGGG LVKPGGSLKL SCAASGFTFS DYGMHWVRQA PEKGLEWVAYISSGSYTIYY TDTVKGRFTI SRDNAKNTLF LQMTSLRSED TAMYYCARRG YGSFYEYYFDYWGQGTTLTV SS;

and/or light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 131) QIVLTQSPAL MSASPGEKVT MTCSASSSVS YMYWYQQKPR SSPKPWIYLTSNLASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQW SSNPFTFGSG TKLEIK.

In an embodiment, the targeting moiety comprises a VHH directed againstPD-1 as disclosed, for example, in U.S. Pat. No. 8,907,065 and WO2008/071447, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the VHHs against PD-1 compriseSEQ ID NOS: 347-351 of U.S. Pat. No. 8,907,065:

SEQ ID No: 347 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 132):EVQLVESGGGLVQAGKSLRLSCAASGSIFSIHAMGWFRQAPGKEREFVAAITWSGGITYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADR AESSWYDYWGQGTQVTVSS;SEQ ID No: 348 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 133):EVQLVESGGGLVQAGGSLRLSCAASGSIASIHAMGWFRQAPGKEREFVAVITWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAGDK HQSSWYDYWGQGTQVTVSS;SEQ ID No: 349 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 134):EVQLVESGGGLVQAGGSLRLSCAASGSISSIHAMGWFRQAPGKEREFVAAITWSGGITYYADSLKGRFTISRDNAKNTGYLQMNSLKPEDTAIYYCAADR AQSSWYDYWGQGTQVTVSS;SEQ ID No: 350 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 135):EVQLVESGGGLVQAGGSLGLSCAASGSIFSINAMAWFRQAPGKEREFVALISWSGGSTYYEDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAADR VDSNWYDYWGQGTQVTVSS;SEQ ID No: 351 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 136):EVQLVESGGGLVQAGGSLRLSCAASGRAFSSGTMGWFRRAPGKEREFVASIPWSGGRIYYADSVKGRFTISRDNAQNTVYLQMNSLKPEDTAVYYCAVKE RSTGWDFASWGQCTQVTVSS.

In an embodiment, the targeting moiety comprises any one of theanti-PD-1 antibodies, or fragments thereof, as disclosed inUS2011/0271358 and WO2010/036959, the entire contents of which arehereby incorporated by reference. In illustrative embodiments, theantibody or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID NOS: 25-29 of US2011/0271358:

SEQ ID No: 25 of US2011/0271358 (SEQ ID NO: 137):QVQLVQSGAELKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDSAVYYCARWRDSSGYHAMDYWGQGTSVTVSS; SEQ ID No: 26 of US2011/0271358 (SEQ ID NO: 138):QVQLVQSGAEVKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYY3/d10CARWRDSSGYHAMDYWGQGTSVTVSS; SEQ ID No: 27 of US2011/0271358 (SEQ ID NO:139): QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVKQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS; SEQ ID No: 28 of US2011/0271358 (SEQ ID NO: 140):QVQLVQSGHEVKQPGASVKMSCKASGYSFTSSWIHWVRQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATLTADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS; SEQ ID No: 29 of US2011/0271358 (SEQ ID NO: 141):QVQLVQSGHEVKQPGASVKVSCKASGYSFTSSWIHWVRQAPGQGLEWIGYIYPSTGFTEYNQKFKDRATITADKSTSTAYMELSSLRSEDTAVYYCARWRDSSGYHAMDYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID NOS: 30-33 of US2011/0271358:

SEQ ID No: 30 of US2011/0271358 (SEQ ID NO: 142):DIVLTQSPASLTLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEEEDFATYYCQHSWEIPY TFGQGTKLEIK; SEQ IDNo: 31 of US2011/0271358 (SEQ ID NO: 143):DIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSWEIPY TFGQGTKLEIK; SEQ IDNo: 32 of US2011/0271358 (SEQ ID NO: 144):EIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFATYYCQHSWEIPY TFGQGTKLEIK; SEQ IDNo: 33 of US2011/0271358 (SEQ ID NO: 145):DIVLTQSPATLSLSPGQRLTISCRASQSVSTSGYSYMHWYQQKPDQSPKLLIKFGSNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSWEIPY TFGQGTKLEIK.

In various embodiments, the present chimeric protein comprises one ormore antibodies directed against PD-1, or antibody fragments thereof,selected from TSR-042 (Tesaro, Inc.), REGN2810 (RegeneronPharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals), and BGB-A317(BeiGene Ltd.)

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against PD-L1.

In some embodiments, the chimeric protein has one or more targetingmoieties which selectively bind a PD-L1 polypeptide. In someembodiments, the chimeric protein comprises one or more antibodies,antibody derivatives or formats, peptides or polypeptides, or fusionproteins that selectively bind a PD-L1 polypeptide.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyMED14736 (aka durvalumab), or fragments thereof. MED14736 is selectivefor PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80receptors. MED14736 and antigen-binding fragments thereof for use in themethods provided herein comprises a heavy chain and a light chain or aheavy chain variable region and a light chain variable region. Thesequence of MED14736 is disclosed in WO/2016/06272, the entire contentsof which are hereby incorporated by reference. In illustrativeembodiments, MED14736 or an antigen-binding fragment thereof for use inthe methods provided herein comprises a heavy chain comprising the aminoacid sequence of:

(SEQ ID NO: 146) EVQLVESGGG LVQPGGSLRL SCAASGFTFS RYWMSWVRQA PGKGLEWVANIKQDGSEKYY VDSVKGRFTI SRDNAKNSLY LQMNSLRAED TAVYYCAREG GWFGELAFDYWGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV SWNSGALTSGVHTFPAVLQS SGLYSLSSVV TVPSSSLGTQ TYICNVNHKP SNTKVDKRVE PKSCDKTHTCPPCPAPEFEG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHNAKTKPREEQY NSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPASIEKTI SKAKGQPREPQVYTLPPSRE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFLYSKLTVDKSR WQQGNVFSCS VMHEALHNHY TQKSLSLSPG K;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 147) EIVLTQSPGT LSLSPGERAT LSCRASQRVS SSYLAWYQQK PGQAPRLLIYDASSRATGIP DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QYGSLPWTFG QGTKVEIKRTVAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSKDSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC.

In illustrative embodiments, the MED14736 or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO:4 ofWO/2016/06272 (SEQ ID NO: 148):

EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NO:3 of WO/2016/06272 (SEQ ID NO: 149):

EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTKVEIK

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyatezolizumab (aka MPDL3280A, RG7446), or fragments thereof. Inillustrative embodiments, atezolizumab or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising the amino acid sequence of:

(SEQ ID NO: 150) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 151) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyavelumab (aka MSB0010718C), or fragments thereof. In illustrativeembodiments, avelumab or an antigen-binding fragment thereof for use inthe methods provided herein comprises a heavy chain comprising the aminoacid sequence of:

(SEQ ID NO: 152) EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYIMMWVRQA PGKGLEWVSSIYPSGGITFY ADTVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCARIK LGTVTTVDYWGQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGVHTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP KSCDKTHTCPPCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNAKTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQVYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLYSKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 153) QSALTQPASV SGSPGQSITI SCTGTSSDVG GYNYVSWYQQ HPGKAPKLMIYDVSNRPSGV SNRFSGSKSG NTASLTISGL QAEDEADYYC SSYTSSSTRV FGTGTKVTVLGQPKANPTVT LFPPSSEELQ ANKATLVCLI SDFYPGAVTV AWKADGSPVK AGVETTKPSKQSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibodyBMS-936559 (aka 12A4, MDX-1105), or fragments thereof, as disclosed inUS 2013/0309250 and WO2007/005874, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments,BMS-936559 or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain variable region comprising theamino acid sequence of:

(SEQ ID NO: 154) QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 155) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3G10, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 3G10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 156) QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYGFSWVRQAPGQGLEWMGWITAYNGNTNYAQKLQGRVTMTTDTSTSTVYMELRSLRSDDTAVYYCARDY FYGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 157) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLVWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPRTFGQ GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody10A5, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 10A5 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 158) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDVHWVRQAPGQRLEWMGWLHADTGITKFSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARER IQLWFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 159) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody5F8, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 5F8 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 160) QVQLVQSGAEVKKPGSSVKVSCKVSGGIFSTYAINWVRQAPGQGLEWMGGIIPIFGTANHAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDQ GIAAALFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 161) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody10H10, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 10H10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 162) EVQLVESGGGLVQPGRSLRLSCAVSGFTFDDYVVHWVRQAPGKGLEWVSGISGNSGNIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAVPF DYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 163) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQ GTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1B12, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 1B12 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 164) QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSSYAISWVRQAPGQGLEWMGGIIPIFGRAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 165) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody7H1, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 7H1 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 166) QVQLVQSGAEVKKPGSSVKVSCKTSGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTTTAYMELSSLRSEDTAVYYCARKYDYVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 167) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody11E6, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 11E6 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 168) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAINWVRQAPGQGLEWMGGIIPIFGSANYAQKFQDRVTITADESTSAAYMELSSLRSEDTAVYYCARDSSGWSRYYMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 169) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPFGGG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody12B7, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 12B7 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 170) QVQLVQSGAEVKEPGSSVKVSCKASGGTFNSYAISWVRQAPGQGLEWMGGIIPLFGIAHYAQKFQGRVTITADESTNTAYMDLSSLRSEDTAVYYCARKYSYVSGSPFGMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 171) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQG TRLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody13G4, or fragments thereof, as disclosed in US 2013/0309250 andWO2007/005874, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 13G4 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 172) EVQLVESGGGLVQPGRSLRLSCAASGITFDDYGMHWVRQAPGKGLEWVSGISWNRGRIEYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGRFRYFDWFLDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 173) AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPFTFGP GTKVDIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1E12, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1E12 or an antigen-binding fragment thereoffor use in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 174) EVKLQESGPS LVKPSQTLSL TCSVTGYSIT SDYWNWIRKF PGNKLEYVGYISYTGSTYYN PSLKSRISIT RDTSKNQYYL QLNSVTSEDT ATYYCARYGG WLSPFDYWGQGTTLTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 175) DIVMTQSHKL MSTSVGDRVS ITCKASQDVG TAVAWYQQKP GQSPKLLIYWASTRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQQ DSSYPLTFGA GTKVELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody1F4, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 1F4 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 176) EVQLQESGPG LVAPSQSLSI TCTVSGFSLT TYSINWIRQP PGKGLEWLGVMWAGGGTNSN SVLKSRLIIS KDNSKSQVFL KMNSLQTDDT ARYYCARYYG NSPYYAIDYWGQGTSVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 177) DIVTTQSHKL MSTSVGDRVS ITCKASQDVG TAVAWYQQKP GQSPKLLIYWASTRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQQ DSSYPLTFGA GTKVELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2G11, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 2G11 or an antigen-binding fragment thereoffor use in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 178) EVKLQESGPS LVKPSQTLSL TCSVTGYSII SDYWNWIRKF PGNKLEYLGYISYTGSTYYN PSLKSRISIT RDTSKNQYYL QLNSVTTEDT ATYYCARRGG WLLPFDYWGQGTTLTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 179) DIVMTQSPSS LAVSVGEKVS MGCKSSQSLL YSSNQKNSLA WYQQKPGQSPKLLIDWASTR ESGVPDRFTG SGSGTDFTLT ISSVKAEDLA VYYCQQYYGY PLTFGAGTKL ELK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3B6, or fragments thereof, as disclosed in US 2014/0044738, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 3B6 or an antigen-binding fragment thereof foruse in the methods provided herein comprises a heavy chain variableregion comprising the amino acid sequence of:

(SEQ ID NO: 180) EVKLQESGPS LVKPGASVKL SCKASGYTFT SYDINWVKQR PGQGLEWIGWIFPRDNNTKY NENFKGKATL TVDTSSTTAY MELHSLTSED SAVYFCTKEN WVGDFDYWGQGTTLTLSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 181) DIVMTQSPAI MSASPGEKVT MTCSASSSIR YMHWYQQKPG TSPKRWISDTSKLTSGVPAR FSGSGSGTSY ALTISSMEAE DAATYYCHQR SSYPWTFGGG TKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3D10, or fragments thereof, as disclosed in US 2014/0044738 andWO2012/145493, the entire disclosures of which are hereby incorporatedby reference. In illustrative embodiments, 3D10 or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of:

(SEQ ID NO: 182) EVQLQQSGPD LVTPGASVRI SCQASGYTFP DYYMNWVKQS HGKSLEWIGDIDPNYGGTTY NQKFKGKAIL TVDRSSSTAY MELRSLTSED SAVYYCARGA LTDWGQGTSL TVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 183) QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIYWFQQKPG SSPKPWIYATFNLASGVPAR FSGSGSGTSY SLTISRVETE DAATYYCQQW SNNPLTFGAG TKLELK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in US2011/0271358 and WO2010/036959, theentire contents of which are hereby incorporated by reference. Inillustrative embodiments, the antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising an amino acid sequence selected from SEQ ID Nos: 34-38 ofUS2011/0271358:

SEQ ID No: 34 of US2011/0271358 (SEQ ID NO: 184):EVQLVQSGPELKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDSAVYYCAR QAWGYPWGQGTLVTVSS; SEQID No: 35 of US2011/0271358 (SEQ ID NO: 185):EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVKQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCAR QAWGYPWGQGTLVTVSS; SEQID No: 36 of US2011/0271358 (SEQ ID NO: 186):EVQLVQSGAEVKKPGASVKMSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCAR QAWGYPWGQGTLVTVSS; SEQID No: 37 of US2011/0271358 (SEQ ID NO: 187):EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATLTSDKSTSTAYMELSSLRSEDTAVYYCAR QAWGYPWGQGTLVTVSS; SEQID No: 38 of US2011/0271358 (SEQ ID NO: 188):EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYVNPFNDGTKYNEMFKGRATITSDKSTSTAYMELSSLRSEDTAVYYCAR QAWGYPWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 39-42 of US2011/0271358:

SEQ ID No: 39 of US2011/0271358 (SEQ ID NO: 189):DIVLTQSPASLALSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEEEDAAMYFCQQSRRV PYTFGQGTKLEIK; SEQ IDNo: 40 of US2011/0271358 (SEQ ID NO: 190):DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAAMYFCQQSRRV PYTFGQGTKLEIK; SEQ IDNo: 41 of US2011/0271358 (SEQ ID NO: 191):EIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAAMYFCQQSRRV PYTFGQGTKLEIK; SEQ IDNo: 42 of US2011/0271358 (SEQ ID NO: 192):DIVLTQSPATLSLSPGERATLSCRATESVEYYGTSLVQWYQQKPGQPPKLLIYAASSVDSGVPSRFSGSGSGTDFTLTINSLEAEDAATYFCQQSRRV PYTFGQGTKLEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.7A4, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.7A4 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 2 of WO 2011/066389 (SEQ ID NO: 193):EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSSISSSGDYIYYADSVKGRFTISRDNAKNSLFLQMNSLKAEDTAVYYCARDLVTSMVAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 7 of WO 2011/066389 (SEQ ID NO: 194):SYELTQPPSVSVSPGQAARITCSGDALPQKYVFWYQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDRSGNHRV FGGGTRLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.9D10, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.9D10 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 12 of WO 2011/066389 (SEQ ID NO: 195):EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGGEQYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWNYGYYDMDVWGQGTTVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 17 of WO 2011/066389 (SEQ ID NO: 196):EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWFQQKPGQAPRLLIFGTSSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSIFT FGPGTKVDIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.14H9, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.14H9 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 22 of WO 2011/066389 (SEQ ID NO: 197):EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 27 of WO 2011/066389 (SEQ ID NO: 198):EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWT FGQGTEVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.20A8, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 2.20A8 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 32 of WO 2011/066389 (SEQ ID NO: 199):EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSAIRGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLHYDSSGYLDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 37 of WO 2011/066389 (SEQ ID NO: 200):DIQMTQSPSSVSASVGDRVTITCRASQGIRSWLAWYQQKPGKAPKLLIYAISRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPLTFGG GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3.15G8, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 3.15G8 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 42 of WO 2011/066389 (SEQ ID NO: 201):EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGGEKYYVDSVKGRFTISRDNAKNSLFLQMNSLRAEDTAVYYCARVQ LYSDYFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 47 of WO 2011/066389 (SEQ ID NO: 202):DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKSGKAPKLLIYAASGLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSHSLPPTFGQ GTKVEIK.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody3.18G1, or fragments thereof, as disclosed in WO 2011/066389, U.S. Pat.No. 8,779,108, and US2014/0356353, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments, 3.18G1 oran antigen-binding fragment thereof for use in the methods providedherein comprises a heavy chain variable region comprising the amino acidsequence of:

SEQ ID No: 52 of WO 2011/066389 (SEQ ID NO: 203):EVQLLESGGDLVQPGGSLRLSCAASGFTFNSYAMSWVRQAPGKGLEWVSTISGSGGFTFSADSVKGRFTISRDNSKNTLFLQMNSLRVEDSAVYSCAKVLVGFNNGCWDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 57 of WO 2011/066389 (SEQ ID NO: 204):SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSNDHVVFG GGTKLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.7A4OPT, or fragments thereof, as disclosed in WO 2011/066389, U.S.Pat. No. 8,779,108, and US2014/0356353, and US2014/0356353, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, 2.7A4OPT or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chainvariable region comprising the amino acid sequence of:

SEQ ID No: 62 of WO 2011/066389 (SEQ ID NO: 205):EVQLVESGGGLVKPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSSISSSGDYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDL VTSMVAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 67 of WO 2011/066389 (SEQ ID NO: 206):SYELTQPPSVSVSPGQTARITCSGDALPQKYVFWYQQKSGQAPVLVIYEDSKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDRSGNHRVFG GGTKLTVL.

In an embodiment, the targeting moiety comprises the anti-PD-L1 antibody2.14H9OPT, or fragments thereof, as disclosed in WO 2011/066389, U.S.Pat. No. 8,779,108, and US2014/0356353, the entire disclosures of whichare hereby incorporated by reference. In illustrative embodiments,2.14H9OPT or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain variable region comprising theamino acid sequence of:

SEQ ID No: 72 of WO 2011/066389 (SEQ ID NO: 207):EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 77 of WO 2011/066389 (SEQ ID NO: 208):EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFG QGTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2016/061142, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 18, 30, 38, 46, 50, 54, 62, 70, and78 of WO2016/061142:

SEQ ID No: 18 of WO2016/061142 (SEQ ID NO: 209):QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWVRQATGQGLEWMGRIDPNSGSTKYNEKFKNRFTISRDDSKNTAYLQMNSLKTEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 30 of WO2016/061142 (SEQ ID NO: 210):EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWVRQATGQGLEWMGRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 38 of WO2016/061142 (SEQ ID NO: 211):EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWVRQAPGQGLEWMGRIDPNSGSTKYNEKFKNRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 46 of WO2016/061142 (SEQ ID NO: 212):EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWIRQSPSRGLEWLGRIDPNSGSTKYNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 50 of WO2016/061142 (SEQ ID NO: 213):EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWIRQPPGKGLEWIGRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 54 of WO2016/061142 (SEQ ID NO: 214):QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMYWIRQSPSRGLEWLGRIDPNSGSTKYNEKFKNRFTISRDDSKNTAYLQMNSLKTEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 62 of WO2016/061142: (SEQ ID NO: 215)EVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMYWVRQARGQRLEWIGRIDPNSGSTKYNEKFKNRLTISKDTSKNQVVLTMTNMDPVDTATYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 70 of WO2016/061142 (SEQ ID NO: 216):QITLKESGPTLVKPTQTLTLTCTFSGYTFTSYWMYWVRQAPGKGLEWVSRIDPNSGSTKYNEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;SEQ ID No: 78 of WO2016/061142 (SEQ ID NO: 217):EVQLVQSGAEVKKPGATVKISCKVSGYTFTSYWMYWVRQARGQRLEWIGRIDPNSGSTKYNEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDY RKGLYAMDYWGQGTTVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO2016/061142:

SEQ ID No: 22 of WO2016/061142 (SEQ ID NO: 218):DIVMTQTPLSLPVTPGEPASISCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:26 of WO2016/061142 (SEQ ID NO: 219):DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:34 of WO2016/061142 (SEQ ID NO: 220):EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:42 of WO2016/061142 (SEQ ID NO: 221):EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNSYPLTFGQ GTKVEIK. SEQ ID No:58 of WO2016/061142 (SEQ ID NO: 222):EIVLTQSPATLSLSPGERATLSCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:66 of WO2016/061142 (SEQ ID NO: 223):DVVMTQSPLSLPVTLGQPASISCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:74 of WO2016/061142 (SEQ ID NO: 224):DIQMTQSPSSLSASVGDRVTITCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:82 of WO2016/061142 (SEQ ID NO: 225):AIQLTQSPSSLSASVGDRVTITCKASQDVGTAVAWYLQKPGQSPQLLIYWASTRHTGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQYNSYPLTFGQ GTKVEIK; SEQ ID No:86 of WO2016/061142 (SEQ ID NO: 226):EIVLTQSPDFQSVTPKEKVTITCKASQDVGTAVAWYQQKPGQAPRLLIYWASTRHTGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPLTFGQ GTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2016/022630, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38,42, and 46 of WO2016/022630:

SEQ ID No: 2 of WO2016/022630 (SEQ ID NO: 227):EVKLVESGGGLVKPGGSLKLSCAASGFIFRSYGMSWVRQTPEKRLEWVASISSGGSTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYDCARGYD SGFAYWGQGTLVTVSE; SEQID No: 6 of WO2016/022630 (SEQ ID NO: 228):EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWVRQTPEKRLEWVASISSGGTTYYPDSVKGRFIISRDNARNILYLQMSSLRSEDTAMYYCAKGYD SGFAYWGQGTLVIVSA; SEQID No: 10 of WO2016/022630 (SEQ ID NO: 229):QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWRGVTTDYNAAFMSRLTITKDNSKSQVFFKMNSLQANDTAIYYCARLGF YAMDYWGQGTSVTVSS; SEQID No: 14 of WO2016/022630 (SEQ ID NO: 230):QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGVTDYNAAFISRLSISKDNSKSQVFFKMNSLQANDTAIYYCARLGF YAMDYWGQGTSVTVSS; SEQID No: 18 of WO2016/022630 (SEQ ID NO: 231):EVKLFESGGGLVQPGGSLKLSCVASGFDFSTYWMHWVRQAPGQGLEWIGQINPDSTTINYAPSLKDRFIISRDNAKNTLFLQMSKVRSEDTALYYCAKPG DYGYDFDCWGQGTTLTVSS;SEQ ID No: 22 of WO2016/022630 (SEQ ID NO: 232):EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYISYSGSTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARSLL WFSTGFAYWGQGTLVTVSA;SEQ ID No: 26 of WO2016/022630 (SEQ ID NO: 233):QVQLKQSGPGLVQPSQSLSITCTVSGFSLTSYGVHWVRQSPGKGLEWLGVIWSGGITDYNAAFKSRLSISKDNSKSQVFFKMNSLQANDTAIYFCARLGF YAMDYWGQGTSVTVSS; SEQID No: 30 of WO2016/022630 (SEQ ID NO: 234):EVKLVESGGGLVKPGGSLKLSCAASGFTFRSYGMSWARQIPEKRLEWVASISSGGTTYYLGSVQGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYD AGFAYWGQGTLVSVSE; SEQID No: 34 of WO2016/022630 (SEQ ID NO: 235):EVQLQESGPSLVKPSQTLSLTCSVTGDSITSGYWTWIRKFPGNKLEYMGYISYTGSTYYNPSLKSRISISRDTSKSQYYLQLNSVTTEDTATYYCARQRD WLGFAYWGQGTLVTVSA;SEQ ID No: 38 of WO2016/022630 (SEQ ID NO: 236):EEKLVESGGGLVKPGGSLKLSCAASGFSFSSYGMSWVRQTPEKRLEWVASISSGGSIYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARGYD AGFAFWGQGTLVTASA; SEQID No: 42 of WO2016/022630 (SEQ ID NO: 237):QITLKESGPTLVKPTQTLTLTCTVSGFSLSTYGVHWIRQPPGKALEWLGVIWRGVTTDYNAAFMSRLTITKDNSKNQVVLTMNNMDPVDTATYYCARLGF YAMDYWGQGTLVTVSS; SEQID No: 46 of WO2016/022630 (SEQ ID NO: 238):EVQLVESGGGLVKPGGSLRLSCAASGFIFRSYGMSWVRQAPGKGLEWVASISSGGSTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYDCARGYD SGFAYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 ofWO2016/022630:

SEQ ID No: 4 of WO2016/022630 (SEQ ID NO: 239):DIVLTQSPASLAVSLGQRATISCRASQSVSTSSSSFMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIKR; SEQ IDNo: 8 of WO2016/022630 (SEQ ID NO: 240):DIVLTQSPPSLAVSLGQRATISCRASQSVSTSSSSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIK; SEQ IDNo: 12 of WO2016/022630 (SEQ ID NO: 241):SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVAWYQQKPGQSPKLLIYYAANRYTGVPDRFTGSGYGTDFTFTISIVQAEDLAVYFCQQDYTSPYTFGG GTKLEIK; SEQ ID No:16 of WO2016/022630 (SEQ ID NO: 242):SIVMTQTPKFLLVSAGDRVTITCKASQSVSNDVGWYQQKPGQSPKLLIYYASNRYSGVPDRFTGSGYGTDFTFTISTVQAEDLAVYFCQQDYTSPYTFGG GTKLEIK; SEQ ID No:20 of WO2016/022630 (SEQ ID NO: 243):DVLMTQTPLYLPVSLGDQASISCRSSQIIVHSNANTYLEWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP YTFGGGTKLEIK; SEQ IDNo: 24 of WO2016/022630 (SEQ ID NO: 244):QIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWNQQKPGSSPKVWIYNTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWRSYPPTLG AGTKLELK; SEQ ID No:28 of WO2016/022630 (SEQ ID NO: 245):QIVLTQSPAIMSASPGEKVTMTCSANSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMGAEDAATYYCQQWSSNPWTFGGG TKLEIK; SEQ ID No: 32of WO2016/022630 (SEQ ID NO: 246):DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQNSWEIPY TFGGGTKLEIK; SEQ IDNo: 36 of WO2016/022630 (SEQ ID NO: 247):DIVMTQTPSSLAVSLGEKVTMSCKSSQSLLYSSNQKNSLAWYQQKPGQSPKLLIYWASNRESGVPDRFTGSSSGTDFTLTISSVKAEDLAVYYCQQYYSY PLTFGAGTKLELK; SEQ IDNo: 40 of WO2016/022630 (SEQ ID NO: 248):DIVLTQSPASLAVSLGQRATISCRASQSVSTSSYSYVHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLNIHPVEEEDTATYYCQHSWEIPY TFGGGTKLEIK; SEQ IDNo: 44 of WO2016/022630 (SEQ ID NO: 249):DIQMTQSPSSLSASVGDRVTITCKASQSVSNDVAWYQQKPGKAPKLLIYYAANRYTGVPDRFSGSGYGTDFTFTISSLQPEDIATYFCQQDYTSPYTFGQ GTKLEIK; SEQ ID No:48 of WO2016/022630 (SEQ ID NO: 250):DIVLTQSPASLAVSPGQRATITCRASQSVSTSSSSFMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTLTINPVEANDTANYYCQHSWEIPY TFGQGTKLEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO2015/112900, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 38, 50, 82, and 86 of WO 2015/112900:

SEQ ID No: 38 of WO2015/112900 (SEQ ID NO: 251):EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQATGQGLEWMGNIYPGTGGSNFDEKFKNRVTITADKSTSTAYMELSSLRSEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 50 of WO 2015/112900 (SEQ ID NO: 252):EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 82 of WO 2015/112900 (SEQ ID NO: 253):QVQLVQSGAEVKKPGASVKVSCKASGYTFTTYWMHWIRQSPSRGLEWLGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;SEQ ID No: 86 of WO 2015/112900 (SEQ ID NO: 254):EVQLVQSGAEVKKPGESLRISCKGSGYTFTTYWMHWVRQAPGQGLEWMGNIYPGTGGSNFDEKFKNRFTISRDNSKNTLYLQMNSLRAEDTAVYYCTRWT TGTGAYWGQGTTVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015/112900:

SEQ ID No: 42 of WO2015/112900 (SEQ ID NO: 255):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 46 of WO 2015/112900 (SEQ ID NO: 256):DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 54 of WO 2015/112900 (SEQ ID NO: 257):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 58 of WO 2015/112900 (SEQ ID NO: 258):DIVMTQTPLSLPVTPGEPASISCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 62 of WO 2015/112900 (SEQ ID NO: 259):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 66 of WO 2015/112900 (SEQ ID NO: 260):EIVLTQSPDFQSVTPKEKVTITCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 70 of WO 2015/112900 (SEQ ID NO: 261):EIVLTQSPATLSLSPGERATLSCKSSQSLLDSGNQKNFLTWYQQKPGQAPRLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 74 of WO 2015/112900 (SEQ ID NO: 262):DIQMTQSPSSLSASVGDRVTITCKSSQSLLDSGNQKNFLTWYLQKPGQSPQLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK; SEQ IDNo: 78 of WO 2015/112900 (SEQ ID NO: 263):DVVMTQSPLSLPVTLGQPASISCKSSQSLLDSGNQKNFLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNDYSY PYTFGQGTKVEIK.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies disclosed in WO 2010/077634 and U.S. Pat. No.8,217,149, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the anti-PD-L1 antibody or anantigen-binding fragment thereof for use in the methods provided hereincomprises a heavy chain region comprising the amino acid sequence of:

SEQ ID No: 20 of WO 2010/077634 (SEQ ID NO: 264):EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSA;

and/or a light chain variable region comprising the amino acid sequenceof:

SEQ ID No: 21 of WO 2010/077634 (SEQ ID NO: 265):DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L1 antibodies obtainable from the hybridoma accessible underCNCM deposit numbers CNCM 1-4122, CNCM 1-4080 and CNCM 1-4081 asdisclosed in US 20120039906, the entire disclosures of which are herebyincorporated by reference.

In an embodiment, the targeting moiety comprises a VHH directed againstPD-L1 as disclosed, for example, in U.S. Pat. No. 8,907,065 and WO2008/071447, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the VHHs against PD-L1 compriseSEQ ID NOS: 394-399 of U.S. Pat. No. 8,907,065:

SEQ ID No: 394 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 266):EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREWASSISSSDGSTYYADSVKGRFTISRDNAKNTVFLQMNSLKPEDTAVYSCAASQAPITIATMMKPFYDYWGQGTQVTVSS; SEQ ID No: 395 of U.S. Pat. No. 8,907,065(SEQ ID NO: 267): EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAKCWFRQAPGKEREWVSCISSSDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAARHGGPLTVEYFFDYWGQGTQVTVSS: SEQ ID No: 396 of U.S. Pat. No. 8,907,065 (SEQID NO: 268): EVQLVESGGGLVQPGGSLRLSCAASGFTFDYYAIGWFRQAPGKAREGVSCISGGDNSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCATGGWKYCSGYDPEYIYWGQGTQVTVSS; SEQ ID No: 397 of U.S. Pat. No. 8,907,065 (SEQID NO: 269): EVQLVESGGGLVQAGGSLRLSCAASGSTFSQYDVGWYRQAPGKQRELVAFSSSGGRTIYPDSVKGRFTFSRDNTKNTVYLQMTSLKPEDTAVYYCKIDW YLNSYWGQGTQVTVSS; SEQID No: 398 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 270):EVQLVESGGGLVQAGGSLRLSCAASGVDASNSAMGWYRQAPGKQREWVARITGGGLIAYTDSVKGRFTISRDNAKSTVYLQMNSLEPEDTAVYYCNTINS RDGWGQGTQVTVSS; SEQID No: 399 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 271):EVQLVESGGGLVQAGGSLTISCAASGITFSDSIVSWYRRARGKQREWVAGISNGGTTKYAESVLGRFTISRDNAKNNVYLQMNGLNPEDTAVYLCKVRQY WGQGTQVTVSS.

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against PD-L2. In some embodiments, thechimeric protein has one or more targeting moieties which selectivelybind a PD-L2 polypeptide. In some embodiments, the chimeric proteincomprises one or more antibodies, antibody derivatives or formats,peptides or polypeptides, or fusion proteins that selectively bind aPD-L2 polypeptide.

In an embodiment, the targeting moiety comprises a VHH directed againstPD-L2 as disclosed, for example, in U.S. Pat. No. 8,907,065 and WO2008/071447, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, the VHHs against PD-1 compriseSEQ ID Nos: 449-455 of U.S. Pat. No. 8,907,065:

SEQ ID No: 449 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 272):EVQLVESGGGLVQAGGSLRLSCAASESTVLINAMGWYRQAPGKQRELVASISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNADVYPQDYGLGYVEGKVYYGHDYWGTGTLVTVSS; SEQ ID No: 450 of U.S. Pat. No.8,907,065 (SEQ ID NO: 273):EVQLVESGGGLVQAGGSLRLSCAASGSTFSNYVSNYAMGWGRQAPGTQRELVASISNGDTTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCFEHQVAGLTWGQGTQVTVSS; SEQ ID No: 451 of U.S. Pat. No. 8,907,065 (SEQ IDNO: 274): EVQLVESGGGLVQAGGSLRLSCVASGXALKIXVMGWYRQAPGKQRELVAAITSGGRTNYSDSVKGRFTISGDNAXNTVYLQMNSLKSEDTAVYYCRE WNSGYPPVDYWGQGTQVTVSS;SEQ ID No: 452 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 275):EVQLVESGGGLVQAGGSLRLSCAASGRTFSSGTMGWFRRAPGKEREFVASIPWSGGRTYYADSVKDRFTISRDNAQNTVFLQMNSLKPEDTAVYYCAFKERSTGWDFASWGQGIQVTVSS; SEQ ID No: 453 of U.S. Pat. No. 8,907,065 (SEQID NO: 276): EVQLVESGGGLVQTGGSLRLSCAASGFTLDYYGIGWFRQAPGKEREGVSFISGSDGSTYYAESVKGRFTISRDKAKNTVYLQMNSLKPEDTAVYYCAADPWGPPSIATMTSYEYKHWGQGTQVTVSS; SEQ ID No: 454 of U.S. Pat. No. 8,907,065(SEQ ID NO: 277): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYTMIWLRRAPGKGFEWVSTIDKDGNTNYVDSVKGRFAVSRDNTKNTLYLQMNSLKPEDTAMYYCTK HGSSARGQGTRVTVSS; SEQID No: 455 of U.S. Pat. No. 8,907,065 (SEQ ID NO: 278):EVQLVESGGGLVEPGGSLRLSCVASGFTFSSYDMSWVRQAPGKGLEWVSTINSGGGITYRGSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYY CENGGSSYRRGQGTQVTVSS.

In an embodiment, the targeting moiety comprises any one of theanti-PD-L2 antibodies disclosed in US2011/0271358 and WO2010/036959, theentire contents of which are hereby incorporated by reference. Inillustrative embodiments, the antibody or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising an amino acid sequence selected from SEQ ID Nos: 43-47 ofUS2011/0271358:

SEQ ID No: 43 of US2011/0271358 (SEQ ID NO: 279):QVQLVQSGAELKKPGASVKMSCKASGYTFTGYTMHWVKQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDSAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 44 of US2011/0271358 (SEQ ID NO: 280):QVQLVQSGAEVKKPGASVKMSCKASGYTFTGYTMHWVKQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 45 of US2011/0271358 (SEQ ID NO: 281):QVQLVQSGAEVKKPGASVKMSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 46 of US2011/0271358 (SEQ ID NO: 282):QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTLTADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS; SEQID No: 47 of US2011/0271358 (SEQ ID NO: 283):QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYTMHWVRQAPGQGLEWIGYINPRSGYTEYNQKFKDRTTITADKSTSTAYMELSSLRSEDTAVYYCARPW FAYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 48-51 of US2011/0271358:

SEQ ID No: 48 of US2011/0271358 (SEQ ID NO: 284):DIVMTQSPASLTVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK; SEQ IDNo: 49 of US2011/0271358 (SEQ ID NO: 285):DIVMTQSPASLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK; SEQ IDNo: 50 of US2011/0271358 (SEQ ID NO: 286):DIVMTQSPAFLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK; SEQ IDNo: 51 of US2011/0271358 (SEQ ID NO: 287):DIVMTQSPAFLSVTPGEKVTITCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSY PLTFGQGTKLEIK.

In various embodiments, the targeting moieties of the invention maycomprise a sequence that targets PD-1, PD-L1, and/or PD-L2 which is atleast about 60%, at least about 61%, at least about 62%, at least about63%, at least about 64%, at least about 65%, at least about 66%, atleast about 67%, at least about 68%, at least about 69%, at least about70%, at least about 71%, at least about 72%, at least about 73%, atleast about 74%, at least about 75%, at least about 76%, at least about77%, at least about 78%, at least about 79%, at least about 80%, atleast about 81%, at least about 82%, at least about 83%, at least about84%, at least about 85%, at least about 86%, at least about 87%, atleast about 88%, at least about 89%, at least about 90%, at least about91%, at least about 92%, at least about 93%, at least about 94%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or 100% identical to any of the sequencesdisclosed herein (e.g. about 60%, or about 61%, or about 62%, or about63%, or about 64%, or about 65%, or about 66%, or about 67%, or about68%, or about 69%, or about 70%, or about 71%, or about 72%, or about73%, or about 74%, or about 75%, or about 76%, or about 77%, or about78%, or about 79%, or about 80%, or about 81%, or about 82%, or about83%, or about 84%, or about 85%, or about 86%, or about 87%, or about88%, or about 89%, or about 90%, or about 91%, or about 92%, or about93%, or about 94%, or about 95%, or about 96%, or about 97%, or about98%, about 99% or about 100% sequence identity with any of the sequencesdisclosed herein).

In various embodiments, the targeting moieties of the invention maycomprise any combination of heavy chain, light chain, heavy chainvariable region, light chain variable region, complementaritydetermining region (CDR), and framework region sequences that targetPD-1, PD-L1, and/or PD-L2 as disclosed herein.

Additional antibodies, antibody derivatives or formats, peptides orpolypeptides, or fusion proteins that selectively bind or target PD-1,PD-L1 and/or PD-L2 are disclosed in WO 2011/066389, US 2008/0025980, US2013/0034559, U.S. Pat. No. 8,779,108, US 2014/0356353, U.S. Pat. No.8,609,089, US 2010/028330, US 2012/0114649, WO 2010/027827, WO2011,/066342, U.S. Pat. No. 8,907,065, WO 2016/062722, WO 2009/101611,WO2010/027827, WO 2011/066342, WO 2007/005874, WO 2001/014556,US2011/0271358, WO 2010/036959, WO 2010/077634, U.S. Pat. No. 8,217,149,US 2012/0039906, WO 2012/145493, US 2011/0318373, U.S. Pat. No.8,779,108, US 20140044738, WO 2009/089149, WO 2007/00587, WO 2016061142,WO 2016,02263, WO 2010/077634, and WO 2015/112900, the entiredisclosures of which are hereby incorporated by reference.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toCD8 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents describedherein. In an embodiment, the present chimeric protein has a targetingmoiety directed against CD8 on T cells and a second targeting moietydirected against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toCD4 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents describedherein. In an embodiment, the present chimeric protein has a targetingmoiety directed against CD4 on T cells and a second targeting moietydirected against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toCD3, CXCR3, CCR4, CCR9, CD70, CD103, or one or more immune checkpointmarkers and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein. In an embodiment, the present chimeric protein has a targetingmoiety directed against CD3 on T cells and a second targeting moietydirected against PD-L1 or PD-L2 on tumor cells.

In some embodiments, the present chimeric protein has one or moretargeting moieties directed against CD3 expressed on T cells. In someembodiments, the chimeric protein has one or more targeting moietieswhich selectively bind a CD3 polypeptide. In some embodiments, thechimeric protein comprises one or more antibodies, antibody derivativesor formats, peptides or polypeptides, or fusion proteins thatselectively bind a CD3 polypeptide.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodymuromonab-CD3 (aka Orthoclone OKT3), or fragments thereof. Muromonab-CD3is disclosed in U.S. Pat. No. 4,361,549 and Wilde et al. (1996)51:865-894, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, muromonab-CD3 or anantigen-binding fragment thereof for use in the methods provided hereincomprises a heavy chain comprising the amino acid sequence of:

(SEQ ID NO: 288) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGGTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 289 QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRADTAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTS TSPIVKSFNRNEC.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyotelixizumab, or fragments thereof. Otelixizumab is disclosed in U.S.Patent Publication No. 20160000916 and Chatenoud et al. (2012)9:372-381, the entire disclosures of which are hereby incorporated byreference. In illustrative embodiments, otelixizumab or anantigen-binding fragment thereof for use in the methods provided hereincomprises a heavy chain comprising the amino acid sequence of:

(SEQ ID NO: 290) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFPMAWVRQAPGKGLEWVSTISTSGGRTYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKFRQYSGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 291) DIQLTQPNSVSTSLGSTVKLSCTLSSGNIENNYVHWYQLYEGRSPTTMIYDDDKRPDGVPDRFSGSIDRSSNSAFLTIHNVAIEDEAIYFCHSYVSSFNVFGGGTKLTVLRQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECS.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyteplizumab (AKA MGA031 and hOKT3γ1(Ala-Ala)), or fragments thereof.Teplizumab is disclosed in Chatenoud et al. (2012) 9:372-381, the entiredisclosures of which are hereby incorporated by reference. Inillustrative embodiments, teplizumab or an antigen-binding fragmentthereof for use in the methods provided herein comprises a heavy chaincomprising the amino acid sequence of:

(SEQ ID NO: 292) QVQLVQSGGGVVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNSKNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK;

and/or a light chain comprising the amino acid sequence of:

(SEQ ID NO: 293) DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL SSPVTKSFNRGEC.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyvisilizumab (AKA Nuvion®; HuM291), or fragments thereof. Visilizumab isdisclosed in U.S. Pat. No. 5,834,597 and WO2004052397, and Cole et al.,Transplantation (1999) 68:563-571, the entire disclosures of which arehereby incorporated by reference. In illustrative embodiments,visilizumab or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain variable regioncomprising the amino acid sequence of:

(SEQ ID NO: 294) QVQLVQSGAEVKKPGASVKVSCKASGYTFISYTMHWVRQAPGQGLEWMGYINPRSGYTHYNQKLKDKATLTADKSASTAYMELSSLRSEDTAVYYCARSA YYDYDGFAYWGQGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof:

(SEQ ID NO: 295) DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQKPGKAPKRLIYDTSKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWSSNPPTFGGG TKVEIK.

In an embodiment, the targeting moiety comprises the anti-CD3 antibodyforalumab (aka NI-0401), or fragments thereof. In various embodiments,the targeting moiety comprises any one of the anti-CD3 antibodiesdisclosed in US20140193399, U.S. Pat. No. 7,728,114, US20100183554, andU.S. Pat. No. 8,551,478, the entire disclosures of which are herebyincorporated by reference.

In illustrative embodiments, the anti-CD3 antibody or an antigen-bindingfragment thereof for use in the methods provided herein comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNos: 2 and 6 of U.S. Pat. No. 7,728,114:

SEQ ID No: 2 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 296):QVQLVESGGGVVQPGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARQM GYWHFDLWGRGTLVTVSS;SEQ ID No: 6 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 297):QVQLVQSGGGVVQSGRSLRLSCAASGFKFSGYGMHWVRQAPGKGLEWVAVIWYDGSKKYYVDSVKGRFTISRDNSKNTLYLQMNSLRGEDTAVYYCARQM GYWHFDLWGRGTLVTVSS;

and/or a light chain variable region comprising the amino acid sequenceof SEQ ID NOs 4 and 8 of U.S. Pat. No. 7,728,114:

SEQ ID No: 4 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 298):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFG GGTKVEIK; SEQ ID No:8 of U.S. Pat. No. 7,728,114 (SEQ ID NO: 299):EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPLTFG GGTKVEIK;

In an embodiment, the targeting moiety comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO:2 of U.S. Pat.No. 7,728,114 and a light chain variable region comprising the aminoacid sequence of SEQ ID NO:4 of U.S. Pat. No. 7,728,114. In anembodiment, the targeting moiety comprises any one of the anti-CD3antibodies disclosed in US2016/0168247, the entire contents of which arehereby incorporated by reference. In illustrative embodiments, theantibody or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 6-9 of US2016/0168247:

SEQ ID No: 6 of US2016/0168247 (SEQ ID NO: 300):EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 7 of US2016/0168247 (SEQ ID NO:301): EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNNLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 8 of US2016/0168247 (SEQ ID NO:302): EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 9 of US2016/0168247 (SEQ ID NO:303): EVKLVESGGGLVKPGRSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSILYLQMNSLKTEDTAMYYCVRHGNFGNSYVSWFAYWGQGTMVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 10-12 of US2016/0168247:

SEQ ID No: 10 of US2016/0168247 (SEQ ID NO: 304):QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALWYSNLWVF GGGTKLTVL; SEQ ID No:11 of US2016/0168247 (SEQ ID NO: 305):QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQADDESIYFCALWYSNLWVF GGGTKLTVL; SEQ ID No:12 of US2016/0168247 (SEQ ID NO: 306):QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQTPGQAFRGLIGGTNKRAPGVPARFSGSILGNKAALTITGAQADDESDYYCALWYSNLW VFGGGTKLTVL.

In an embodiment, the targeting moiety comprises any one of the anti-CD3antibodies disclosed in US2015/0175699, the entire contents of which arehereby incorporated by reference. In illustrative embodiments, theantibody or an antigen-binding fragment thereof for use in the methodsprovided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID No: 9 of US2015/0175699:

SEQ ID No: 9 of US2015/0175699 (SEQ ID NO: 307):QVQLVQSGSELKKPGASVKMSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKFKDRATLTTDKSTSTAYMQLSSLRSEDTAVYYCAR YYDDHYSLDYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID No: 10 of US2015/0175699:

SEQ ID No: 10 of US2015/0175699 (SEQ ID NO: 308):QIVLTQSPATLSLSPGERATMSCSASSSVSYMNWYQQKPGKAPKRWIYDTSKLASGVPSRFRGSGSGTDYTLTISSLQPEDFATYYCQQWSSNPFTFG GGTKVEIK.

In an embodiment, the targeting moiety comprises any one of the anti-CD3antibodies disclosed in U.S. Pat. No. 8,784,821, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 2, 18, 34, 50, 66, 82, 98 and 114 ofU.S. Pat. No. 8,784,821:

SEQ ID No: 2 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 309):ELQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARLSPYCTNGVCWDAFDIWGQGTMVTVSS; SEQ ID No: 18 of U.S. Pat. No. 8,784,821 (SEQID NO: 310): ELQLVESGGGLVKPGRSLRLSCTASGFTFGDYAMSWFRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTPQLWLLQDAFDIWGQGTMVTVSS; SEQ ID No: 34 of U.S. Pat. No. 8,784,821 (SEQ IDNO: 311): ELQLVESGPGLVKPSGTLSLTCAVSGGSISSRNWWSWVRQPPGKGLEWIGDIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCASGY TSCRDAFDIWGQGTMVTVSS;SEQ ID No: 50 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 312):ELQLVEWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGRGRFLGWLLGGSNWFDPWGQGTLVTVSS; SEQ ID No: 66 of U.S. Pat. No. 8,784,821(SEQ ID NO: 313): ELQLVEWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGPD RMGHGFDIWGQGTMVTVSS;SEQ ID No: 82 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 314):ELQLVESGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARDRRRIAARQYYGMDVWGQGTTVTVSS; SEQ ID No: 98 of U.S. Pat. No. 8,784,821(SEQ ID NO: 315): ELQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMGWVRQAPGKGLEWVSAVSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAKFLGHYYGMDVWGQGTTVTVSS; SEQ ID No: 114 of U.S. Pat. No. 8,784,821 (SEQ IDNO: 316): ELQLVESGPVLVKPTDTLTLTCTVSGFSLNNPRMGVSWIRQPPGKTLEWLAHIFPSDAKAHSASLKSRLTISKDTSKSQVVPTMTNMDPVDTATYYCARILGEYYPPAWFDPWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 10, 26, 42, 58, 74, 90, 106 and 122 of U.S. Pat. No. 8,784,821:

SEQ ID No: 10 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 317):ELQMTQSPSSLSASVGDRVSITCRASQTISNYLNWYQLKPGKAPKLLIYAASTLQSEVPTRFSGSGSGTDFTLTISGLHPEDFATYYCQQFNSYPRTFGQ GTKVEIK; SEQ ID No:26 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 318):ELQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQ GTKLEIK; SEQ ID No:42 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 319):ELVMTQSPSSLSASVGDRVTITCRASQGIGNYLAWYQQKPGQPPKMLIYWASIRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSNPQTFGQ GTKVEIK; SEQ ID No:58 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 320):ELVMTQSPSSLSASVGDRVTITCRASQGISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQ GTKVDIK; SEQ ID No:74 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 321):ELQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKSGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSSPWTFGQ GTKVEIK; SEQ ID No:90 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 322):ELVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPLTFG GGTKVEIK; SEQ ID No:106 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 323):ELQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNAYPYTFGQ GTKVEIK; SEQ ID No:122 of U.S. Pat. No. 8,784,821 (SEQ ID NO: 324):ELVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQY LKIPYTFGQGTKVEIK.

In an embodiment, the targeting moiety comprises any one of the anti-CD3binding constructs disclosed in US20150118252, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 6 and 86 of US20150118252:

SEQ ID No: 6 of US20150118252 (SEQ ID NO: 325):QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTMTTDTSISTAYMELSRLRSDDTAVYYCARYY DDHYCLDYWGQGTLVTVSS;SEQ ID No: 86 of US20150118252 (SEQ ID NO: 326):QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYTMHWVRQAPGQGLEWMGYINPSRGYTNYNQKFKDRVTMTTDTSISTAYMELSRLRSDDTAVYYCARYY DDHYSLDYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID No: 3 of US2015/0175699:

SEQ ID No: 3 of US20150118252 (SEQ ID NO: 327):EIVLTQSPATLSLSPGERATLSCSASSSVSYMNWYQQKPGQAPRLLIYDTSKLASGVPAHFRGSGSGTDYTLTISSLEPEDFAVYYCQQWSSNPFTFGQG TKVEIK.

In an embodiment, the targeting moiety comprises any one of the anti-CD3binding proteins disclosed in US2016/0039934, the entire contents ofwhich are hereby incorporated by reference. In illustrative embodiments,the antibody or an antigen-binding fragment thereof for use in themethods provided herein comprises a heavy chain comprising an amino acidsequence selected from SEQ ID Nos: 6-9 of US2016/0039934:

SEQ ID No: 6 of US2016/0039934 (SEQ ID NO: 328):EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 7 of US2016/0039934 (SEQ ID NO:329): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSS; SEQ ID No: 8 of US2016/0039934 (SEQ ID NO:330): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSYFAYWGQGTLVTVSS; SEQ ID No: 9 of US2016/0039934 (SEQ ID NO:331): EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSHFAYWGQGTLVTVSS;

and/or a light chain comprising an amino acid sequence selected from SEQID Nos: 1-4 of US2016/0039934:

SEQ ID No: 1 of US2016/0039934 (SEQ ID NO: 332):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKGLIGGTNKRAPGVPSRFSGSLIGDKATLTISSLQPEDFATYYCALWYSNLWV FGQGTKVEIK; SEQ IDNo: 2 of US2016/0039934 (SEQ ID NO: 333):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKGLIGGTNKRAPGVPARFSGSGSGTDFTLTISSLQPEDFATYYCALWYSNLWV FGQGTKVEIK; SEQ IDNo: 3 of US2016/0039934 (SEQ ID NO: 334):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKALIGGTNKRAPGVPSRFSGSLIGDKATLTISSLQPEDFATYYCALWYSNLWV FGQGTKVEIK; SEQ IDNo: 4 of US2016/0039934 (SEQ ID NO: 335):DIQMTQSPSSLSASVGDRVTITCRSSTGAVTTSNYANWVQQKPGKAPKGLIGGTNKRAPGVPSRFSGSLIGDKATLTISSLQPEDFATYYCALWYSNLW VFGQGTKVEIK;

In various embodiments, the targeting moieties of the invention maycomprise a sequence that targets CD3 which is at least about 60%, atleast about 61%, at least about 62%, at least about 63%, at least about64%, at least about 65%, at least about 66%, at least about 67%, atleast about 68%, at least about 69%, at least about 70%, at least about71%, at least about 72%, at least about 73%, at least about 74%, atleast about 75%, at least about 76%, at least about 77%, at least about78%, at least about 79%, at least about 80%, at least about 81%, atleast about 82%, at least about 83%, at least about 84%, at least about85%, at least about 86%, at least about 87%, at least about 88%, atleast about 89%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or 100% identical to any of the sequences disclosed herein (e.g.about 60%, or about 61%, or about 62%, or about 63%, or about 64%, orabout 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, about 99% or about100% sequence identity with any of the sequences disclosed herein).

In various embodiments, the targeting moieties of the invention maycomprise any combination of heavy chain, light chain, heavy chainvariable region, light chain variable region, complementaritydetermining region (CDR), and framework region sequences that target CD3as disclosed herein. In various embodiments, the targeting moieties ofthe invention may comprise any heavy chain, light chain, heavy chainvariable region, light chain variable region, complementaritydetermining region (CDR), and framework region sequences of theCD3-specific antibodies including, but not limited to, X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2,TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6,T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, WT31 and F101.01. TheseCD3-specific antibodies are well known in the art and, inter alia,described in Tunnacliffe (1989), Int. Immunol. 1, 546-550, the entiredisclosures of which are hereby incorporated by reference.

Additional antibodies, antibody derivatives or formats, peptides orpolypeptides, or fusion proteins that selectively bind or target CD3 aredisclosed in US Patent Publication No. 2016/0000916, U.S. Pat. Nos.4,361,549, 5,834,597, 6,491,916, 6,406,696, 6,143,297, 6,750,325 andInternational Publication No. WO 2004/052397, the entire disclosures ofwhich are hereby incorporated by reference.

In some embodiments, the CD3 targeting moiety is a scFv. In someembodiments, the CD3 targeting moiety is:

(SEQ ID NO: 365) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGSTGGGGSGGGGSGGGGSDIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a T cell, for example, mediated by targeting toPD-1 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents describedherein.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a B cell,for example, mediated by targeting to CD10, CD19, CD20, CD21, CD22,CD23, CD24, CD37, CD38, CD39, CD40, CD70, CD72, CD73, CD74, CDw75,CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86,CD89, CD98, CD126, CD127, CDw130, CD138, or CDw150; and (ii) a targetingmoiety is directed against a tumor cell, along with any of the modified(e.g. mutant) signaling agents described herein.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a B cell, for example, mediated by targeting toCD19, CD20 or CD70 and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a B cell, for example, mediated by targeting toCD20 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents describedherein. In an embodiment, the present chimeric protein has a targetingmoiety directed against CD20 on B cells and a second targeting moietydirected against PD-L1 or PD-L2 on tumor cells.

In some embodiments, the CD20 targeting moiety is a scFv. In someembodiments, the CD20 targeting moiety is:

(SEQ ID NO: 366) QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRGSTGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSS.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a NK cell,for example, mediated by targeting to 2B4/SLAMF4, KIR2DS4, CD155/PVR,KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7,LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, Fc-epsilon RII, LMIR6/CD300LE,Fc-γ RI/CD64, MICA, Fc-γ RIIB/CD32b, MICB, Fc-γ RIIC/CD32c, MULT-1, Fc-γRIIA/CD32a, Nectin-2/CD112, Fc-γ RIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C,FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44,Fc-Receptor-like 3/CD16-2, NKp46/NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1,Rae-1 α, Rae-1 β, Rae-1 delta, H60, Rae-1 epsilon, ILT2/CD85j, Rae-1 γ,ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158,TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d, orULBP-3; and (ii) a targeting moiety is directed against a tumor cell,along with any of the modified (e.g. mutant) signaling agents describedherein.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a NK cell, for example, mediated by targeting toKid alpha, DNAM-1 or CD64 and (ii) a targeting moiety is directedagainst a tumor cell, along with any of the modified (e.g. mutant)signaling agents described herein.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a NK cell, for example, mediated by targeting toKIR1 and (ii) a targeting moiety is directed against a tumor cell, alongwith any of the modified (e.g. mutant) signaling agents describedherein. In an embodiment, the present chimeric protein has a targetingmoiety directed against KIR1 on NK cells and a second targeting moietydirected against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a NK cell, for example, mediated by targeting toTIGIT or KIR1 and (ii) a targeting moiety is directed against a tumorcell, along with any of the modified (e.g. mutant) signaling agentsdescribed herein. In an embodiment, the present chimeric protein has atargeting moiety directed against TIGIT on NK cells and a secondtargeting moiety directed against PD-L1 or PD-L2 on tumor cells.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against a dendriticcell, for example, mediated by targeting to CLEC-9A, XCR1, RANK,CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-A1/MSR, CD5L, SREC-1,CL-PI/COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2,TLR6, TLR3, TLR9, 4-IBB Ligand/TNFSF9, IL-12/IL-23 p40,4-Amino-1,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k,8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, lutegrin α 4/CD49d, Aag,Integrin β 2/CD18, AMICA, Langerin, B7-2/CD86, Leukotriene B4 RI, B7-H3,LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93, LMIR3/CD300LF,CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4-a,CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5,MMR, CD97, NCAMLI, CD2F-10/SLAMF9, Osteoactivin GPNMB, Chern 23, PD-L2,CLEC-1, RP105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33,DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7,DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F,Dectin-2/CLEC6A, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM,EMMPRIN/CD147, TCCR/1/1/SX-1, Fc-γ R1/CD64, TLR3, Fc-γ RIIB/CD32b,TREM-1, Fc-γ RIIC/CD32c, TREM-2, Fc-γ RIIA/CD32a, TREM-3, Fc-γRIII/CD16, TREML1/TLT-1, ICAM-2/CD102, or Vanilloid R1; and (ii) atargeting moiety is directed against a tumor cell, along with any of themodified (e.g. mutant) signaling agents described herein.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to CLEC-9A, DC-SIGN, CD64, CLEC4A, or DEC205 and (ii) atargeting moiety is directed against a tumor cell, along with any of themodified (e.g. mutant) signaling agents described herein. In anembodiment, the present chimeric protein has a targeting moiety directedagainst CLEC9A on dendritic cells and a second targeting moiety directedagainst PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to CLEC9A and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein. In an embodiment, the present chimeric proteinhas a targeting moiety directed against CLEC9A on dendritic cells and asecond targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to XCR1 and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein. In an embodiment, the present chimeric proteinhas a targeting moiety directed against XCR1 on dendritic cells and asecond targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a dendritic cell, for example, mediated bytargeting to RANK and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein. In an embodiment, the present chimeric proteinhas a targeting moiety directed against RANK on dendritic cells and asecond targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

By way of non-limiting example, in various embodiments, the presentchimeric protein has (i) a targeting moiety directed against amonocyte/macrophage, for example, mediated by targeting to SIRP1a,B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Conmmon β Chain, Integrin α4/CD49d, BLAME/SLAMF8, Integrin α X/CDIIc, CCL6/C10, Integrin β 2/CD18,CD155/PVR, Integrin β 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3,Leukotriene B4 R1, CD40/TNFRSF5, LIMPIIISR-B2, CD43, LMIR1/CD300A, CD45,LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97,LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1,ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB,Fc-γ RI/CD64, Osteopontin, Fc-γ RIIB/CD32b, PD-L2, Fc-γ RIIC/CD32c,Siglec-3/CD33, Fc-γ RIIA/CD32a, SIGNR1/CD209, Fc-γ RIII/CD16, SLAM,GM-CSF R α, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ RI, TLR4, IFN-gannnaR2, TREM-1, IL-I RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k,TREML1/TLT-1, 2B4/SLAMF 4, IL-10 R α, ALCAM, IL-10 R β,AminopeptidaseN/ANPEP, ILT2/CD85j, Common β Chain, ILT3/CD85k, ClqR1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, CD206, Integrin α 4/CD49d,CCR5, Integrin α M/CDII b, CCR8, Integrin α X/CDIIc, CD155/PVR, Integrinβ 2/CD18, CD14, Integrin β 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45,Leukotriene B4-R1, CD68, LIMPIIISR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97,LMIR2/CD300c, CD163, LMIR3/CD300LF, Coagulation Factor III/TissueFactor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1,CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR,Endoglin/CD105, NCAM-L1, Fc-γ RI/CD64, PSGL-1, Fc-γ RIIIICD16, RP105,G-CSF R, L-Selectin, GM-CSF R α, Siglec-3/CD33, HVEM/TNFRSF14, SLAM,ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6 R, TREM-2,CXCRI/IL-8 RA, TREM-3, or TREMLI/TLT-1; and (ii) a targeting moiety isdirected against a tumor cell, along with any of the modified (e.g.mutant) signaling agents described herein.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a monocyte/macrophage, for example, mediated bytargeting to B7-H1, CD31/PECAM-1, CD163, CCR2, or Macrophage MannoseReceptor CD206 and (ii) a targeting moiety is directed against a tumorcell, along with any of the modified (e.g. mutant) signaling agentsdescribed herein.

In one embodiment, the present chimeric protein has (i) a targetingmoiety directed against a monocyte/macrophage, for example, mediated bytargeting to SIRP1a and (ii) a targeting moiety is directed against atumor cell, along with any of the modified (e.g. mutant) signalingagents described herein. In an embodiment, the present chimeric proteinhas a targeting moiety directed against SIRP1a on macrophage cells and asecond targeting moiety directed against PD-L1 or PD-L2 on tumor cells.

In various embodiments, the present chimeric protein has one or moretargeting moieties directed against a checkpoint marker, e.g. one ormore of PD-1/PD-L1 or PD-L2, CD28/CD80 or CD86, CTLA4/CD80 or CD86,ICOS/ICOSL or B7RP1, BTLA/HVEM, KIR, LAG3, CD137/CD137L, OX40/OX40L,CD27, CD40L, TIM3/Gal9, and A2aR. In one embodiment, the presentchimeric protein has (i) a targeting moiety directed against acheckpoint marker on a T cell, for example, PD-1 and (ii) a targetingmoiety directed against a tumor cell, for example, PD-L1 or PD-L2, alongwith any of the modified (e.g. mutant) signaling agents describedherein. In an embodiment, the present chimeric protein has a targetingmoiety directed against PD-1 on T cells and a second targeting moietydirected against PD-L1 on tumor cells. In another embodiment, thepresent chimeric protein has a targeting moiety directed against PD-1 onT cells and a second targeting moiety directed against PD-L2 on tumorcells.

In some embodiments, the present chimeric protein comprises two or moretargeting moieties directed to the same or different immune cells. Insome embodiments, the present chimeric protein has (i) one or moretargeting moieties directed against an immune cell selected from a Tcell, a B cell, a dendritic cell, a macrophage, a NK cell, or subsetsthereof and (ii) one or more targeting moieties directed against eitherthe same or another immune cell selected from a T cell, a B cell, adendritic cell, a macrophage, a NK cell, or subsets thereof, along withany of the modified (e.g. mutant) signaling agents described herein(e.g., modified IFN-β).

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a T cell and one or more targetingmoieties directed against the same or another T cell. In one embodiment,the present chimeric protein comprises one or more targeting moietiesdirected against a T cell and one or more targeting moieties directedagainst a B cell. In one embodiment, the present chimeric proteincomprises one or more targeting moieties directed against a T cell andone or more targeting moieties directed against a dendritic cell. In oneembodiment, the present chimeric protein comprises one or more targetingmoieties against a T cell and one or more targeting moieties directedagainst a macrophage. In one embodiment, the present chimeric proteincomprises one or more targeting moieties against a T cell and one ormore targeting moieties directed against a NK cell. For example, in anillustrative embodiment, the chimeric protein may include a targetingmoiety against CD8 and a targeting moiety against Clec9A. In anotherillustrative embodiment, the chimeric protein may include a targetingmoiety against CD8 and a targeting moiety against CD3. In anotherillustrative embodiment, the chimeric protein may include a targetingmoiety against CD8 and a targeting moiety against PD-1.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a B cell and one or more targetingmoieties directed against the same or another B cell. In one embodiment,the present chimeric protein comprises one or more targeting moietiesdirected against a B cell and one or more targeting moieties directedagainst a T cell. In one embodiment, the present chimeric proteincomprises one or more targeting moieties directed against a B cell andone or more targeting moieties directed against a dendritic cell. In oneembodiment, the present chimeric protein comprises one or more targetingmoieties against a B cell and one or more targeting moieties directedagainst a macrophage. In one embodiment, the present chimeric proteincomprises one or more targeting moieties against a B cell and one ormore targeting moieties directed against a NK cell.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a dendritic cell and one or moretargeting moieties directed against the same or another dendritic cell.In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a dendritic cell and one or moretargeting moieties directed against a T cell. In one embodiment, thepresent chimeric protein comprises one or more targeting moietiesdirected against a dendritic cell and one or more targeting moietiesdirected against a B cell. In one embodiment, the present chimericprotein comprises one or more targeting moieties against a dendriticcell and one or more targeting moieties directed against a macrophage.In one embodiment, the present chimeric protein comprises one or moretargeting moieties against a dendritic cell and one or more targetingmoieties directed against a NK cell.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a macrophage and one or moretargeting moieties directed against the same or another macrophage. Inone embodiment, the present chimeric protein comprises one or moretargeting moieties directed against a macrophage and one or moretargeting moieties directed against a T cell. In one embodiment, thepresent chimeric protein comprises one or more targeting moietiesdirected against a macrophage and one or more targeting moietiesdirected against a B cell. In one embodiment, the present chimericprotein comprises one or more targeting moieties against a macrophageand one or more targeting moieties directed against a dendritic cell. Inone embodiment, the present chimeric protein comprises one or moretargeting moieties against a macrophage and one or more targetingmoieties directed against a NK cell.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties directed against an NK cell and one or more targetingmoieties directed against the same or another NK cell. In oneembodiment, the present chimeric protein comprises one or more targetingmoieties directed against an NK cell and one or more targeting moietiesdirected against a T cell. In one embodiment, the present chimericprotein comprises one or more targeting moieties directed against an NKcell and one or more targeting moieties directed against a B cell.

In one embodiment, the present chimeric protein comprises one or moretargeting moieties against an NK cell and one or more targeting moietiesdirected against a macrophage. In one embodiment, the present chimericprotein comprises one or more targeting moieties against an NK cell andone or more targeting moieties directed against a dendritic cell.

In one embodiment, the present chimeric protein comprises a targetingmoiety directed against a tumor cell and a second targeting moietydirected against the same or a different tumor cell. In suchembodiments, the targeting moieties may bind to any of the tumorantigens described herein.

In various embodiments, the targeting moiety of the invention maycomprise a sequence which has one or more amino acid mutations withrespect to any one of the sequences disclosed herein. In variousembodiments, the targeting moiety comprises a sequence that has about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 or more amino acid mutations with respectto any one of the amino acid sequences of targeting moieties disclosedherein. In some embodiments, the one or more amino acid mutations may beindependently selected from substitutions, insertions, deletions, andtruncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, the substitutions may also include non-classicalamino acids (e.g. selenocysteine, pyrrolysine, N-formylmethionineβ-alanine, GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA),D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu,ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosme, citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

In various embodiments, the amino acid mutation may be in the CDRs ofthe targeting moiety (e.g., the CDR1, CDR2 or CDR3 regions). In anotherembodiment, amino acid alteration may be in the framework regions (FRs)of the targeting moiety (e.g., the FR1, FR2, FR3, or FR4 regions).

Modification of the amino acid sequences may be achieved using any knowntechnique in the art e.g., site-directed mutagenesis or PCR basedmutagenesis. Such techniques are described, for example, in Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., 1989.

Targeting Moiety Formats

In various embodiments, the targeting moiety of the present chimericprotein is a protein-based agent capable of specific binding, such as anantibody or derivatives thereof. In an embodiment, the targeting moietycomprises an antibody. In various embodiments, the antibody is afull-length multimeric protein that includes two heavy chains and twolight chains. Each heavy chain includes one variable region (e.g.,V_(H)) and at least three constant regions (e.g., CH₁, CH₂ and CH₃), andeach light chain includes one variable region (V_(L)) and one constantregion (C_(L)). The variable regions determine the specificity of theantibody. Each variable region comprises three hypervariable regionsalso known as complementarity determining regions (CDRs) flanked by fourrelatively conserved framework regions (FRs). The three CDRs, referredto as CDR1, CDR2, and CDR3, contribute to the antibody bindingspecificity. In some embodiments, the antibody is a chimeric antibody.In some embodiments, the antibody is a humanized antibody.

In some embodiments, the targeting moiety comprises antibody derivativesor formats. In some embodiments, the targeting moiety of the presentchimeric protein is a single-domain antibody, a recombinantheavy-chain-only antibody (VHH), a single-chain antibody (scFv), a sharkheavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein,knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; anAnticalin; an AdNectin; an Affilin; a Microbody; a peptide aptamer; analterase; a plastic antibody; a phylomer; a stradobody; a maxibody; anevibody; a fynomer, an armadillo repeat protein, a Kunitz domain, anavimer, an atrimer, a probody, an immunobody, a triomab, a troybody; apepbody; a vaccibody, a UniBody; Affimers, a DuoBody, a Fv, a Fab, aFab′, a F(ab′)₂, a peptide mimetic molecule, or a synthetic molecule, asdescribed in US Patent Nos. or Patent Publication Nos. U.S. Pat. No.7,417,130, US 2004/132094, U.S. Pat. No. 5,831,012, US 2004/023334, U.S.Pat. Nos. 7,250,297, 6,818,418, US 2004/209243, U.S. Pat. Nos.7,838,629, 7,186,524, 6,004,746, 5,475,096, US 2004/146938, US2004/157209, U.S. Pat. Nos. 6,994,982, 6,794,144, US 2010/239633, U.S.Pat. No. 7,803,907, US 2010/119446, and/or U.S. Pat. No. 7,166,697, thecontents of which are hereby incorporated by reference in theirentireties. See also, Storz MAbs. 2011 May-June; 3(3): 310-317.

In one embodiment, the targeting moiety comprises a single-domainantibody, such as VHH from, for example, an organism that produces VHHantibody such as a camelid, a shark, or a designed VHH. VHHs areantibody-derived therapeutic proteins that contain the unique structuraland functional properties of naturally-occurring heavy-chain antibodies.VHH technology is based on fully functional antibodies from camelidsthat lack light chains. These heavy-chain antibodies contain a singlevariable domain (VHH) and two constant domains (CH2 and CH3). VHHs arecommercially available under the trademark of NANOBODIES.

In some embodiments, the VHH is a humanized VHH or camelized VHH.

In various embodiments, the targeting moiety of the present chimericprotein is a protein-based agent capable of specific binding to a cellreceptor, such as a natural ligand for the cell receptor. In variousembodiments, the cell receptor is found on one or more immune cells,which can include, without limitation, T cells, cytotoxic T lymphocytes,T helper cells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g. M1 macrophages), B cells, dendritic cells,or subsets thereof. In some embodiments, the cell receptor is found onmegakaryocytes, thrombocytes, erythrocytes, mast cells, basophils,neutrophils, eosinophils, or subsets thereof.

In some embodiments, the targeting moiety is a natural ligand such as achemokine. Exemplary chemokines that may be included in the chimericprotein of the invention include, but are not limited to, CCL1, CCL2,CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14,CCL15, CCL16, CL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7,CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16,CXCL17, XCL1, XCL2, CX3CL1, HCC-4, and LDGF-PBP. In an illustrativeembodiment, the targeting moiety may be XCL1 which is a chemokine thatrecognizes and binds to the dendritic cell receptor XCR1. In anotherillustrative embodiment, the targeting moiety is CCL1, which is achemokine that recognizes and binds to CCR8. In another illustrativeembodiment, the targeting moiety is CCL2, which is a chemokine thatrecognizes and binds to CCR2 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL3, which is a chemokine thatrecognizes and binds to CCR1, CCR5, or CCR9. In another illustrativeembodiment, the targeting moiety is CCL4, which is a chemokine thatrecognizes and binds to CCR1 or CCR5 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL5, which is a chemokine thatrecognizes and binds to CCR1 or CCR3 or CCR4 or CCR5. In anotherillustrative embodiment, the targeting moiety is CCL6, which is achemokine that recognizes and binds to CCR1. In another illustrativeembodiment, the targeting moiety is CCL7, which is a chemokine thatrecognizes and binds to CCR2 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL8, which is a chemokine thatrecognizes and binds to CCR1 or CCR2 or CCR2B or CCR5 or CCR9. Inanother illustrative embodiment, the targeting moiety is CCL9, which isa chemokine that recognizes and binds to CCR1. In another illustrativeembodiment, the targeting moiety is CCL10, which is a chemokine thatrecognizes and binds to CCR1. In another illustrative embodiment, thetargeting moiety is CCL11, which is a chemokine that recognizes andbinds to CCR2 or CCR3 or CCR5 or CCR9. In another illustrativeembodiment, the targeting moiety is CCL13, which is a chemokine thatrecognizes and binds to CCR2 or CCR3 or CCR5 or CCR9. In anotherillustrative embodiment, the targeting moiety is CCL14, which is achemokine that recognizes and binds to CCR1 or CCR9. In anotherillustrative embodiment, the targeting moiety is CCL15, which is achemokine that recognizes and binds to CCR1 or CCR3. In anotherillustrative embodiment, the targeting moiety is CCL16, which is achemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. Inanother illustrative embodiment, the targeting moiety is CCL17, which isa chemokine that recognizes and binds to CCR4. In another illustrativeembodiment, the targeting moiety is CCL19, which is a chemokine thatrecognizes and binds to CCR7. In another illustrative embodiment, thetargeting moiety is CCL20, which is a chemokine that recognizes andbinds to CCR6. In another illustrative embodiment, the targeting moietyis CCL21, which is a chemokine that recognizes and binds to CCR7. Inanother illustrative embodiment, the targeting moiety is CCL22, which isa chemokine that recognizes and binds to CCR4. In another illustrativeembodiment, the targeting moiety is CCL23, which is a chemokine thatrecognizes and binds to CCR1. In another illustrative embodiment, thetargeting moiety is CCL24, which is a chemokine that recognizes andbinds to CCR3. In another illustrative embodiment, the targeting moietyis CCL25, which is a chemokine that recognizes and binds to CCR9. Inanother illustrative embodiment, the targeting moiety is CCL26, which isa chemokine that recognizes and binds to CCR3. In another illustrativeembodiment, the targeting moiety is CCL27, which is a chemokine thatrecognizes and binds to CCR10. In another illustrative embodiment, thetargeting moiety is CCL28, which is a chemokine that recognizes andbinds to CCR3 or CCR10. In another illustrative embodiment, thetargeting moiety is CXCL1, which is a chemokine that recognizes andbinds to CXCR1 or CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL2, which is a chemokine that recognizes andbinds to CXCR2. In another illustrative embodiment, the targeting moietyis CXCL3, which is a chemokine that recognizes and binds to CXCR2. Inanother illustrative embodiment, the targeting moiety is CXCL4, which isa chemokine that recognizes and binds to CXCR3B. In another illustrativeembodiment, the targeting moiety is CXCL5, which is a chemokine thatrecognizes and binds to CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL6, which is a chemokine that recognizes andbinds to CXCR1 or CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL8, which is a chemokine that recognizes andbinds to CXCR1 or CXCR2. In another illustrative embodiment, thetargeting moiety is CXCL9, which is a chemokine that recognizes andbinds to CXCR3. In another illustrative embodiment, the targeting moietyis CXCL10, which is a chemokine that recognizes and binds to CXCR3. Inanother illustrative embodiment, the targeting moiety is CXCL11, whichis a chemokine that recognizes and binds to CXCR3 or CXCR7. In anotherillustrative embodiment, the targeting moiety is CXCL12, which is achemokine that recognizes and binds to CXCR4 or CXCR7. In anotherillustrative embodiment, the targeting moiety is CXCL13, which is achemokine that recognizes and binds to CXCR5. In another illustrativeembodiment, the targeting moiety is CXCL16, which is a chemokine thatrecognizes and binds to CXCR6. In another illustrative embodiment, thetargeting moiety is LDGF-PBP, which is a chemokine that recognizes andbinds to CXCR2. In another illustrative embodiment, the targeting moietyis XCL2, which is a chemokine that recognizes and binds to XCR1. Inanother illustrative embodiment, the targeting moiety is CX3CL1, whichis a chemokine that recognizes and binds to CX3CR1.

In various embodiments, the present chimeric protein comprises targetingmoieties in various combinations. In an illustrative embodiment, thepresent chimeric protein may comprise two targeting moieties, whereinboth targeting moieties are antibodies or derivatives thereof. Inanother illustrative embodiment, the present chimeric protein maycomprise two targeting moieties, wherein both targeting moieties arenatural ligands for cell receptors. In a further illustrativeembodiment, the present chimeric protein may comprise two targetingmoieties, wherein one of the targeting moieties is an antibody orderivative thereof, and the other targeting moiety is a natural ligandfor a cell receptor.

In various embodiments, the recognition domain of the present chimericprotein functionally modulates (by way of non-limitation, partially orcompletely neutralizes) the target (e.g. antigen, receptor) of interest,e.g. substantially inhibiting, reducing, or neutralizing a biologicaleffect that the antigen has. For example, various recognition domainsmay be directed against one or more tumor antigens that are activelysuppressing, or have the capacity to suppress, the immune system of, forexample, a patient bearing a tumor. For example, in some embodiments,the present chimeric protein functionally modulates immune inhibitorysignals (e.g. checkpoint inhibitors), for example, one or more of TIM-3,BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3,CD244, CD160, TIGIT, SIRPα, ICOS, CD172a, and TMIGD2. For example, insome embodiments, the present chimeric protein is engineered to disrupt,block, reduce, and/or inhibit the transmission of an immune inhibitorysignal, by way of non-limiting example, the binding of PD-1 with PD-L1or PD-L2 and/or the binding of CTLA-4 with one or more of AP2M1, CD80,CD86, SHP-2, and PPP2R5A.

In various embodiments, the recognition domain of the present chimericprotein binds but does not functionally modulate the target (e.g.antigen, receptor) of interest, e.g. the recognition domain is, or isakin to, a binding antibody. For instance, in various embodiments, therecognition domain simply targets the antigen or receptor but does notsubstantially inhibit, reduce or functionally modulate a biologicaleffect that the antigen or receptor has. For example, some of thesmaller antibody formats described above (e.g. as compared to, forexample, full antibodies) have the ability to target hard to accessepitopes and provide a larger spectrum of specific binding locales. Invarious embodiments, the recognition domain binds an epitope that isphysically separate from an antigen or receptor site that is importantfor its biological activity (e.g. the antigen's active site).

Such non-neutralizing binding finds use in various embodiments of thepresent invention, including methods in which the present chimericprotein is used to directly or indirectly recruit active immune cells toa site of need via an effector antigen, such as any of those describedherein. For example, in various embodiments, the present chimericprotein may be used to directly or indirectly recruit cytotoxic T cellsvia CD8 to a tumor cell in a method of reducing or eliminating a tumor(e.g. the chimeric protein may comprise an anti-CD8 recognition domainand a recognition domain directed against a tumor antigen). In suchembodiments, it is desirable to directly or indirectly recruitCD8-expressing cytotoxic T cells but not to functionally modulate theCD8 activity. On the contrary, in these embodiments, CD8 signaling is animportant piece of the tumor reducing or eliminating effect. By way offurther example, in various methods of reducing or eliminating tumors,the present chimeric protein is used to directly or indirectly recruitdendritic cells (DCs) via CLEC9A (e.g. the chimeric protein may comprisean anti-CLEC9A recognition domain and a recognition domain directedagainst a tumor antigen). In such embodiments, it is desirable todirectly or indirectly recruit CLEC9A-expressing DCs but not tofunctionally modulate the CLEC9A activity. On the contrary, in theseembodiments, CLEC9A signaling is an important piece of the tumorreducing or eliminating effect.

In various embodiments, the recognition domain of the present chimericprotein binds to XCR1 e.g. on dendritic cells. For instance, therecognition domain, in some embodiments comprises all or part of XCL1 ora non-neutralizing anti-XCR1 agent.

In various embodiments, the recognition domain of the present chimericprotein binds to an immune modulatory antigen (e.g. immune stimulatoryor immune inhibitory). In various embodiments, the immune modulatoryantigen is one or more of 4-1BB, OX-40, HVEM, GITR, CD27, CD28, CD30,CD40, ICOS ligand; OX-40 ligand, LIGHT (CD258), GITR ligand, CD70, B7-1,B7-2, CD30 ligand, CD40 ligand, ICOS, ICOS ligand, CD137 ligand andTL1A. In various embodiments, such immune stimulatory antigens areexpressed on a tumor cell. In various embodiments, the recognitiondomain of the present chimeric protein binds but does not functionallymodulate such immune stimulatory antigens and therefore allowsrecruitment of cells expressing these antigens without the reduction orloss of their potential tumor reducing or eliminating capacity.

In various embodiments, the recognition domain of the present chimericprotein may be in the context of chimeric protein that comprises tworecognition domains that have neutralizing activity, or comprises tworecognition domains that have non-neutralizing (e.g. binding) activity,or comprises one recognition domain that has neutralizing activity andone recognition domain that has non-neutralizing (e.g. binding)activity.

Signaling Agents

In one aspect, the present invention provides a chimeric protein thatincludes a signaling agent (for instance, an immune-modulating agent).In various embodiments, the signaling agent is modified to have reducedaffinity or activity for one or more of its receptors, which allows forattenuation of activity (inclusive of agonism or antagonism) and/orprevents non-specific signaling or undesirable sequestration of thechimeric protein.

In various embodiments, the signaling agent is antagonistic in its wildtype form and bears one or more mutations that attenuate itsantagonistic activity. In various embodiments, the signaling agent isantagonistic due to one or more mutations, e.g. an agonistic signalingagent is converted to an antagonistic signaling agent and, such aconverted signaling agent, optionally, also bears one or more mutationsthat attenuate its antagonistic activity (e.g. as described in WO2015/007520, the entire contents of which are hereby incorporated byreference).

In various embodiments, the signaling agent is selected from modifiedversions of cytokines, growth factors, and hormones. Illustrativeexamples of such cytokines, growth factors, and hormones include, butare not limited to, lymphokines, monokines, traditional polypeptidehormones, such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and tumornecrosis factor-β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-α; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II; osteoinductive factors; interferons such as, for example, interferon-α,interferon-β and interferon-γ (and interferon type I, II, and III),colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as, for example, IL-1, IL-1a, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, and IL-18; atumor necrosis factor such as, for example, TNF-α or TNF-6; and otherpolypeptide factors including, for example, LIF and kit ligand (KL). Asused herein, cytokines, growth factors, and hormones include proteinsobtained from natural sources or produced from recombinant bacterial,eukaryotic or mammalian cell culture systems and biologically activeequivalents of the native sequence cytokines.

In some embodiments, the signaling agent is a modified version of agrowth factor selected from, but not limited to, transforming growthfactors (TGFs) such as TGF-α and TGF-β, epidermal growth factor (EGF),insulin-like growth factor such as insulin-like growth factor-I and -II,fibroblast growth factor (FGF), heregulin, platelet-derived growthfactor (PDGF), vascular endothelial growth factor (VEGF).

In an embodiment, the growth factor is a modified version of afibroblast growth factor (FGF). Illustrative FGFs include, but are notlimited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10,FGF11, FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19,FGF20, FGF21, FGF22, and FGF23.

In an embodiment, the growth factor is a modified version of a vascularendothelial growth factor (VEGF). Illustrative VEGFs include, but arenot limited to, VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF and isoformsthereof including the various isoforms of VEGF-A such as VEGF₁₂₁,VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF₁₈₉, and VEGF₂₀₆.

In an embodiment, the growth factor is a modified version of atransforming growth factor (TGF). Illustrative TGFs include, but are notlimited to, TGF-α and TGF-β and subtypes thereof including the varioussubtypes of TGF-β including TGβ31, TGβ2, and TGβ3.

In some embodiments, the signaling agent is a modified version of ahormone selected from, but not limited to, human chorionic gonadotropin,gonadotropin releasing hormone, an androgen, an estrogen,thyroid-stimulating hormone, follicle-stimulating hormone, luteinizinghormone, prolactin, growth hormone, adrenocorticotropic hormone,antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growthhormone releasing hormone, corticotropin-releasing hormone,somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroidhormone, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline,progesterone, insulin, glucagon, amylin, calcitriol, calciferol,atrial-natriuretic peptide, gastrin, secretin, cholecystokinin,neuropeptide Y, ghrelin, PYY3-36, insulin-like growth factor (IGF),leptin, thrombopoietin, erythropoietin (EPO), and angiotensinogen. Insome embodiments, the signaling agent is EPO.

In some embodiments, the signaling agent is an immune-modulating agent,e.g. one or more of an interleukin, interferon, and tumor necrosisfactor.

In some embodiments, the signaling agent is an interleukin or a modifiedinterleukin, including for example IL-1; IL-2; IL-3; IL-4; IL-5; IL-6;IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16;IL-17; IL-18; IL-19; IL- 20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26;IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or afragment, variant, analogue, or family-member thereof. Interleukins area group of multi-functional cytokines synthesized by lymphocytes,monocytes, and macrophages. Known functions include stimulatingproliferation of immune cells (e.g., T helper cells, B cells,eosinophils, and lymphocytes), chemotaxis of neutrophils and Tlymphocytes, and/or inhibition of interferons. Interleukin activity canbe determined using assays known in the art: Matthews et al., inLymphokines and Interferens: A Practical Approach, Clemens et al., eds,IRL Press, Washington, D.C. 1987, pp. 221-225; and Orencole & Dinarello(1989) Cytokine 1, 14-20.

In some embodiments, the signaling agent is an interferon or a modifiedversion of an interferon such as interferon types I, II, and III.Illustrative interferons, including for example, interferon-α-1, 2, 4,5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon-β and interferon-γ,interferon κ, interferon ε, interferon τ, and interferon {circumflexover (ω)}.

In some embodiments, the signaling agent is a tumor necrosis factor(TNF) or a modified version of a tumor necrosis factor (TNF) or aprotein in the TNF family, including but not limited to, TNF-α, TNF-6,LT-β, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

In various embodiments, the signaling agent is a modified (e.g. mutant)form of the signaling agent having one or more mutations. In variousembodiments, the mutations allow for the modified signaling agent tohave one or more of attenuated activity such as one or more of reducedbinding affinity, reduced endogenous activity, and reduced specificbioactivity relative to unmodified or unmutated, i.e. the wild type formof the signaling agent (e.g. comparing the same signaling agent in awild type form versus a modified (e.g. mutant) form). In someembodiments, the mutations which attenuate or reduce binding or affinityinclude those mutations which substantially reduce or ablate binding oractivity. In some embodiments, the mutations which attenuate or reducebinding or affinity are different than those mutations whichsubstantially reduce or ablate binding or activity. Consequentially, invarious embodiments, the mutations allow for the signaling agent to haveimproved safety, e.g. have reduced systemic toxicity, reduced sideeffects, and reduced off-target effects relative to unmutated, i.e. wildtype, signaling agent (e.g. comparing the same signaling agent in a wildtype form versus a modified (e.g. mutant) form).

In various embodiments, the signaling agent is modified to have one ormore mutations that reduce its binding affinity or activity for one ormore of its receptors. In some embodiments, the signaling agent ismodified to have one or more mutations that substantially reduce orablate binding affinity or activity for the receptors. In someembodiments, the activity provided by the wild type signaling agent isagonism at the receptor (e.g. activation of a cellular effect at a siteof therapy). For example, the wild type signaling agent may activate itsreceptor. In such embodiments, the mutations result in the modifiedsignaling agent to have reduced or ablated activating activity at thereceptor. For example, the mutations may result in the modifiedsignaling agent to deliver a reduced activating signal to a target cellor the activating signal could be ablated. In some embodiments, theactivity provided by the wild type signaling agent is antagonism at thereceptor (e.g. blocking or dampening of a cellular effect at a site oftherapy). For example, the wild type signaling agent may antagonize orinhibit the receptor. In these embodiments, the mutations result in themodified signaling agent to have a reduced or ablated antagonizingactivity at the receptor. For example, the mutations may result in themodified signaling agent to deliver a reduced inhibitory signal to atarget cell or the inhibitory signal could be ablated. In variousembodiments, the signaling agent is antagonistic due to one or moremutations, e.g. an agonistic signaling agent is converted to anantagonistic signaling agent (e.g. as described in WO 2015/007520, theentire contents of which are hereby incorporated by reference) and, sucha converted signaling agent, optionally, also bears one or moremutations that reduce its binding affinity or activity for one or moreof its receptors or that substantially reduce or ablate binding affinityor activity for one or more of its receptors.

In some embodiments, the reduced affinity or activity at the receptor isrestorable by attachment with one or more of the targeting moieties. Inother embodiments, the reduced affinity or activity at the receptor isnot substantially restorable by the activity of one or more of thetargeting moieties.

In various embodiments, the chimeric proteins of the present inventionreduce off-target effects because their signaling agents have mutationsthat weaken or ablate binding affinity or activity at a receptor. Invarious embodiments, this reduction in side effects is observed relativewith, for example, the wild type signaling agents. In variousembodiments, the signaling agent is active on target cells because thetargeting moiety(ies) compensates for the missing/insufficient binding(e.g., without limitation and/or avidity) required for substantialactivation. In various embodiments, the modified signaling agent issubstantially inactive en route to the site of therapeutic activity andhas its effect substantially on specifically targeted cell types whichgreatly reduces undesired side effects.

In some embodiments, the signaling agent may include one or moremutations that attenuate or reduce binding or affinity for one receptor(i.e., a therapeutic receptor) and one or more mutations thatsubstantially reduce or ablate binding or activity at a second receptor.In such embodiments, these mutations may be at the same or at differentpositions (i.e., the same mutation or multiple mutations). In someembodiments, the mutation(s) that reduce binding and/or activity at onereceptor is different than the mutation(s) that substantially reduce orablate at another receptor. In some embodiments, the mutation(s) thatreduce binding and/or activity at one receptor is the same as themutation(s) that substantially reduce or ablate at another receptor. Insome embodiments, the present chimeric proteins have a modifiedsignaling agent that has both mutations that attenuate binding and/oractivity at a therapeutic receptor and therefore allow for a morecontrolled, on-target therapeutic effect (e.g. relative wild typesignaling agent) and mutations that substantially reduce or ablatebinding and/or activity at another receptor and therefore reduce sideeffects (e.g. relative to wild type signaling agent).

In some embodiments, the substantial reduction or ablation of binding oractivity is not substantially restorable with a targeting moiety. Insome embodiments, the substantial reduction or ablation of binding oractivity is restorable with a targeting moiety. In various embodiments,substantially reducing or ablating binding or activity at a secondreceptor also may prevent deleterious effects that are mediated by theother receptor. Alternatively, or in addition, substantially reducing orablating binding or activity at the other receptor causes thetherapeutic effect to improve as there is a reduced or eliminatedsequestration of the therapeutic chimeric proteins away from the site oftherapeutic action. For instance, in some embodiments, this obviates theneed of high doses of the present chimeric proteins that compensate forloss at the other receptor. Such ability to reduce dose further providesa lower likelihood of side effects.

In various embodiments, the modified signaling agent comprises one ormore mutations that cause the signaling agent to have reduced,substantially reduced, or ablated affinity, e.g. binding (e.g. K_(D))and/or activation (for instance, when the modified signaling agent is anagonist of its receptor, measurable as, for example, K_(A) and/or EC₅₀)and/or inhibition (for instance, when the modified signaling agent is anantagonist of its receptor, measurable as, for example, K_(I) and/orIC₅₀), for one or more of its receptors. In various embodiments, thereduced affinity at the immumodulating agent's receptor allows forattenuation of activity (inclusive of agonism or antagonism). In suchembodiments, the modified signaling agent has about 1%, or about 3%,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 10%-20%,about 20%-40%, about 50%, about 40%-60%, about 60%-80%, about 80%-100%of the affinity for the receptor relative to the wild type signalingagent. In some embodiments, the binding affinity is at least about2-fold lower, about 3-fold lower, about 4-fold lower, about 5-foldlower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about9-fold lower, at least about 10-fold lower, at least about 15-foldlower, at least about 20-fold lower, at least about 25-fold lower, atleast about 30-fold lower, at least about 35-fold lower, at least about40-fold lower, at least about 45-fold lower, at least about 50-foldlower, at least about 100-fold lower, at least about 150-fold lower, orabout 10-50-fold lower, about 50-100-fold lower, about 100-150-foldlower, about 150-200-fold lower, or more than 200-fold lower relative tothe wild type signaling agent.

In embodiments wherein the chimeric protein has mutations that reducebinding at one receptor and substantially reduce or ablate binding at asecond receptor, the attenuation or reduction in binding affinity of amodified signaling agent for one receptor is less than the substantialreduction or ablation in affinity for the other receptor.

In some embodiments, the attenuation or reduction in binding affinity ofa modified signaling agent for one receptor is less than the substantialreduction or ablation in affinity for the other receptor by about 1%, orabout 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

In various embodiments, substantial reduction or ablation refers to agreater reduction in binding affinity and/or activity than attenuationor reduction.

In various embodiments, the modified signaling agent comprises one ormore mutations that reduce the endogenous activity of the signalingagent to about 75%, or about 70%, or about 60%, or about 50%, or about40%, or about 30%, or about 25%, or about 20%, or about 10%, or about5%, or about 3%, or about 1%, e.g., relative to the wild type signalingagent

In various embodiments, the modified signaling agent comprises one ormore mutations that cause the signaling agent to have reduced affinityand/or activity for a receptor of any one of the cytokines, growthfactors, and hormones as described herein.

In some embodiments, the modified signaling agent comprises one or moremutations that cause the signaling agent to have reduced affinity forits receptor that is lower than the binding affinity of the targetingmoiety(ies) for its(their) receptor(s). In some embodiments, thisbinding affinity differential is between signaling agent/receptor andtargeting moiety/receptor on the same cell. In some embodiments, thisbinding affinity differential allows for the signaling agent, e.g.mutated signaling agent, to have localized, on-target effects and tominimize off-target effects that underlie side effects that are observedwith wild type signaling agent. In some embodiments, this bindingaffinity is at least about 2-fold, or at least about 5-fold, or at leastabout 10-fold, or at least about 15-fold lower, or at least about25-fold, or at least about 50-fold lower, or at least about 100-fold, orat least about 150-fold.

Receptor binding activity may be measured using methods known in theart. For example, affinity and/or binding activity may be assessed byScatchard plot analysis and computer-fitting of binding data (e.g.Scatchard, 1949) or by reflectometric interference spectroscopy underflow through conditions, as described by Brecht et al. (1993), theentire contents of all of which are hereby incorporated by reference.

The amino acid sequences of the wild type signaling agents describedherein are well known in the art. Accordingly, in various embodimentsthe modified signaling agent comprises an amino acid sequence that hasat least about 60%, or at least about 61%, or at least about 62%, or atleast about 63%, or at least about 64%, or at least about 65%, or atleast about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99% sequence identity with the known wild type amino acidsequences of the signaling agents described herein (e.g. about 60%, orabout 61%, or about 62%, or about 63%, or about 64%, or about 65%, orabout 66%, or about 67%, or about 68%, or about 69%, or about 70%, orabout 71%, or about 72%, or about 73%, or about 74%, or about 75%, orabout 76%, or about 77%, or about 78%, or about 79%, or about 80%, orabout 81%, or about 82%, or about 83%, or about 84%, or about 85%, orabout 86%, or about 87%, or about 88%, or about 89%, or about 90%, orabout 91%, or about 92%, or about 93%, or about 94%, or about 95%, orabout 96%, or about 97%, or about 98%, or about 99% sequence identity).

In various embodiments the modified signaling agent comprises an aminoacid sequence that has at least about 60%, or at least about 61%, or atleast about 62%, or at least about 63%, or at least about 64%, or atleast about 65%, or at least about 66%, or at least about 67%, or atleast about 68%, or at least about 69%, or at least about 70%, or atleast about 71%, or at least about 72%, or at least about 73%, or atleast about 74%, or at least about 75%, or at least about 76%, or atleast about 77%, or at least about 78%, or at least about 79%, or atleast about 80%, or at least about 81%, or at least about 82%, or atleast about 83%, or at least about 84%, or at least about 85%, or atleast about 86%, or at least about 87%, or at least about 88%, or atleast about 89%, or at least about 90%, or at least about 91%, or atleast about 92%, or at least about 93%, or at least about 94%, or atleast about 95%, or at least about 96%, or at least about 97%, or atleast about 98%, or at least about 99% sequence identity with any ofamino acid sequences of the signaling agents disclosed herein (e.g.about 60%, or about 61%, or about 62%, or about 63%, or about 64%, orabout 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%, or about 96%, or about 97%, or about 98%, or about 99%sequence identity).

In various embodiments, the modified signaling agent comprises an aminoacid sequence having one or more amino acid mutations. In someembodiments, the one or more amino acid mutations may be independentlyselected from substitutions, insertions, deletions, and truncations.

In some embodiments, the amino acid mutations are amino acidsubstitutions, and may include conservative and/or non-conservativesubstitutions as described elsewhere herein.

As described herein, the modified signaling agents bear mutations thataffect affinity and/or activity at one or more receptors. In variousembodiments, there is reduced affinity and/or activity at a therapeuticreceptor, e.g. a receptor through which a desired therapeutic effect ismediated (e.g. agonism or antagonism). In various embodiments, themodified signaling agents bear mutations that substantially reduce orablate affinity and/or activity at a receptor, e.g. a receptor throughwhich a desired therapeutic effect is not mediated (e.g. as the resultof promiscuity of binding). The receptors of any modified signalingagents, e.g. one of the cytokines, growth factors, and hormones asdescribed herein, are known in the art.

Illustrative mutations which provide reduced affinity and/or activity(e.g. agonistic) at a receptor are found in WO 2013/107791 (e.g. withregard to interferons), WO 2015/007542 (e.g. with regard tointerleukins), and WO 2015/007903 (e.g. with regard to TNF), the entirecontents of each of which are hereby incorporated by reference.Illustrative mutations which provide reduced affinity and/or activity(e.g. antagonistic) at a therapeutic receptor are found in WO2015/007520, the entire contents of which are hereby incorporated byreference.

In some embodiments, the modified signaling agent comprises one or moremutations that cause the signaling agent to have reduced affinity and/oractivity for a type I cytokine receptor, a type II cytokine receptor, achemokine receptor, a receptor in the Tumor Necrosis Factor Receptor(TNFR) superfamily, TGF-beta Receptors, a receptor in the immunoglobulin(Ig) superfamily, and/or a receptor in the tyrosine kinase superfamily.

In various embodiments, the receptor for the signaling agent is a Type Icytokine receptor. Type I cytokine receptors are known in the art andinclude, but are not limited to receptors for IL2 (beta-subunit), IL3,IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, and alsothe receptors for Thrombopoietin (TPO), Prolactin, and Growth hormone.Illustrative type I cytokine receptors include, but are not limited to,GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPOreceptor, and type I IL receptors.

In various embodiments, the receptor for the signaling agent is a TypeII cytokine receptor. Type II cytokine receptors are multimericreceptors composed of heterologous subunits, and are receptors mainlyfor interferons. This family of receptors includes, but is not limitedto, receptors for interferon-α, interferon-β and interferon-γ, IL10,IL22, and tissue factor. Illustrative type II cytokine receptorsinclude, but are not limited to, IFN-α receptor (e.g. IFNAR1 andIFNAR2), IFN-β receptor, IFN-γ receptor (e.g. IFNGR1 and IFNGR2), andtype II IL receptors.

In various embodiments, the receptor for the signaling agent is a Gprotein-coupled receptor. Chemokine receptors are G protein-coupledreceptors with seven transmembrane structure and coupled to G-proteinfor signal transduction. Chemokine receptors include, but are notlimited to, CC chemokine receptors, CXC chemokine receptors, CX3Cchemokine receptors, and XC chemokine receptor (XCR1). Exemplarychemokine receptors include, but are not limited to, CCR1, CCR2, CCR3,CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B,CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

In various embodiments, the receptor for the signaling agent is a TNFRfamily member. Tumor necrosis factor receptor (TNFR) family membersshare a cysteine-rich domain (CRD) formed of three disulfide bondssurrounding a core motif of CXXCXXC creating an elongated molecule.Exemplary tumor necrosis factor receptor family members include: CDI 20a(TNFRSFIA), CD 120b (TNFRSFIB), Lymphotoxin beta receptor (LTBR,TNFRSF3), CD 134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6),TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137(TNFRSF9), TNFRSFIOA (TNFRSFIOA), TNFRSFIOB, (TNFRSFIOB), TNFRSFIOC(TNFRSFIOC), TNFRSFIOD (TNFRSFIOD), RANK (TNFRSFI IA), Osteoprotegerin(TNFRSFI IB), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C(TNFRSF13C), TNFRSF14 (TNFRSF14), Nerve growth factor receptor (NGFR,TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19(TNFRSF19), TNFRSF21 (TNFRSF21), and TNFRSF25 (TNFRSF25).

In various embodiments, the receptor for the signaling agent is aTGF-beta receptor. TGF-beta receptors are single pass serine/threoninekinase receptors. TGF-beta receptors include, but are not limited to,TGFBR1, TGFBR2, and TGFBR3.

In various embodiments, the receptor for the signaling agent is an Igsuperfamily receptor. Receptors in the immunoglobulin (Ig) superfamilyshare structural homology with immunoglobulins. Receptors in the Igsuperfamily include, but are not limited to, interleukin-1 receptors,CSF-1R, PDGFR (e.g. PDGFRA and PDGFRB), and SCFR.

In various embodiments, the receptor for the signaling agent is atyrosine kinase superfamily receptor. Receptors in the tyrosine kinasesuperfamily are well known in the art. There are about 58 known receptortyrosine kinases (RTKs), grouped into 20 subfamilies. Receptors in thetyrosine kinase superfamily include, but are not limited to, FGFreceptors and their various isoforms such as FGFR1, FGFR2, FGFR3, FGFR4,and FGFR5.

In an embodiment, the modified signaling agent is interferon α. In suchembodiments, the modified IFN-α agent has reduced affinity and/oractivity for the IFN-α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2chains. In some embodiments, the modified IFN-α agent has substantiallyreduced or ablated affinity and/or activity for the IFN-α/β receptor(IFNAR), i.e., IFNAR1 and/or IFNAR2 chains.

Mutant forms of interferon α are known to the person skilled in the art.In an illustrative embodiment, the modified signaling agent is theallelic form IFN-α2a having the amino acid sequence of:

IFN-α2a (SEQ ID NO: 336):CDLPQTHSLGSRRTLMLLAQMRKISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMR SFSLSTNLQESLRSKE

In an illustrative embodiment, the modified signaling agent is theallelic form IFN-α2b having the amino acid sequence of (which differsfrom IFN-α2a at amino acid position 23):

IFN-α2b (SEQ ID NO: 337):CDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRS FSLSTNLQESLRSKE

In some embodiments, said IFN-α2 mutant (IFN-α2a or IFN-α2b) is mutatedat one or more amino acids at positions 144-154, such as amino acidpositions 148, 149 and/or 153. In some embodiments, the IFN-α2 mutantcomprises one or more mutations selected from L153A, R149A, and M148A.Such mutants are described, for example, in WO2013/107791 and Piehler etal., (2000) J. Biol. Chem, 275:40425-33, the entire contents of all ofwhich are hereby incorporated by reference.

In some embodiments, the IFN-α2 mutants have reduced affinity and/oractivity for IFNAR1. In some embodiments, the IFN-α2 mutant comprisesone or more mutations selected from F64A, N65A, T69A, L80A, Y85A, andY89A, as described in WO2010/030671, the entire contents of which ishereby incorporated by reference.

In some embodiments, the IFN-α2 mutant comprises one or more mutationsselected from K133A, R144A, R149A, and L153A as described inWO2008/124086, the entire contents of which is hereby incorporated byreference.

In some embodiments, the IFN-α2 mutant comprises one or more mutationsselected from R120E and R120E/K121E, as described in WO2015/007520 andWO2010/030671, the entire contents of which are hereby incorporated byreference. In such embodiments, said IFN-α2 mutant antagonizes wild typeIFN-α2 activity. In such embodiments, said mutant IFN-α2 has reducedaffinity and/or activity for IFNAR1 while affinity and/or activity ofIFNR2 is retained.

In some embodiments, the human IFN-α2 mutant comprises (1) one or moremutations selected from R120E and R120E/K121E, which, without wishing tobe bound by theory, create an antagonistic effect and (2) one or moremutations selected from K133A, R144A, R149A, and L153A, which, withoutwishing to be bound by theory, allow for an attenuated effect at, forexample, IFNAR2. In an embodiment, the human IFN-α2 mutant comprisesR120E and L153A.

In some embodiments, the human IFN-α2 mutant comprises one or moremutations selected from, L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V,K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A,R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A, and N156Aas disclosed in WO 2013/059885, the entire disclosures of which arehereby incorporated by reference. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or L30A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or R33A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or M148A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations H57Y, E58N, Q61S, and/or L153A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations N65A, L80A, Y85A, and/or Y89A asdisclosed in WO 2013/059885. In some embodiments, the human IFN-α2mutant comprises the mutations N65A, L80A, Y85A, Y89A, and/or D114A asdisclosed in WO 2013/059885.

In an embodiment, the modified signaling agent is interferon β. In suchembodiments, the modified interferon β agent has reduced affinity and/oractivity for the IFN-α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2chains. In some embodiments, the modified interferon β agent hassubstantially reduced or ablated affinity and/or activity for theIFN-α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains.

In an embodiment, the modified signaling agent is interferon β. In suchembodiments, the modified interferon β agent has reduced affinity and/oractivity for the interferon-gamma receptor (IFNGR), i.e., IFNGR1 andIFNGR2 chains. In some embodiments, the modified interferon β agent hassubstantially reduced or ablated affinity and/or activity for theinterferon-gamma receptor (IFNGR), i.e., IFNGR1 and/or IFNGR2 chains.

In some embodiments, the modified signaling agent is vascularendothelial growth factor (VEGF). VEGF is a potent growth factor thatplays major roles in physiological but also pathological angiogenesis,regulates vascular permeability and can act as a growth factor on cellsexpressing VEGF receptors. Additional functions include, among others,stimulation of cell migration in macrophage lineage and endothelialcells. Several members of the VEGF family of growth factors exist, aswell as at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3).Members of the VEGF family can bind and activate more than one VEGFRtype. For example, VEGF-A binds VEGFR-1 and -2, while VEGF-C can bindVEGFR-2 and -3. VEGFR-1 and -2 activation regulates angiogenesis whileVEGFR-3 activation is associated with lymphangiogenesis. The majorpro-angiogenic signal is generated from activation of VEGFR-2. VEGFR-1activation has been reported to be possibly associated with negativerole in angiogenesis. It has also been reported that VEGFR-1 signalingis important for progression of tumors in vivo via bone marrow-derivedVEGFR-1 positive cells (contributing to formation of premetastatic nichein the bone). Several therapies based on VEGF-A directed/neutralizingtherapeutic antibodies have been developed, primarily for use intreatment of various human tumors relying on angiogenesis. These are notwithout side effects though. This may not be surprising considering thatthese operate as general, non-cell/tissue specific VEGF/VEGFRinteraction inhibitors. Hence, it would be desirable to restrict VEGF(e.g. VEGF-A)/VEGFR-2 inhibition to specific target cells (e.g. tumorvasculature endothelial cells).

In some embodiments, the VEGF is VEGF-A, VEGF-B, VEFG-C, VEGF-D, orVEGF-E and isoforms thereof including the various isoforms of VEGF-Asuch as VEGF₁₂₁, VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF₁₈₉, andVEGF₂₀₆. In some embodiments, the modified signaling agent has reducedaffinity and/or activity for VEGFR-1 (Flt-1) and/or VEGFR-2 (KDR/Flk-1).In some embodiments, the modified signaling agent has substantiallyreduced or ablated affinity and/or activity for VEGFR-1 (Flt-1) and/orVEGFR-2 (KDR/Flk-1). In an embodiment, the modified signaling agent hasreduced affinity and/or activity for VEGFR-2 (KDR/Flk-1) and/orsubstantially reduced or ablated affinity and/or activity for VEGFR-1(Flt-1). Such an embodiment finds use, for example, in wound healingmethods or treatment of ischmia-related diseases (without wishing to bebound by theory, mediated by VEGFR-2's effects on endothelial cellfunction and angiogenesis). In various embodiments, binding to VEGFR-1(Flt-1), which is linked to cancers and pro-inflammatory activities, isavoided. In various embodiments, VEGFR-1 (Flt-1) acts a decoy receptorand therefore substantially reduces or ablates affinity at this receptoravoids sequestration of the therapeutic agent. In an embodiment, themodified signaling agent has substantially reduced or ablated affinityand/or activity for VEGFR-1 (Flt-1) and/or substantially reduced orablated affinity and/or activity for VEGFR-2 (KDR/Flk-1). In someembodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, themodified signaling agent has reduced affinity and/or activity forVEGFR-3. Alternatively, the modified signaling agent has substantiallyreduced or ablated affinity and/or activity for VEGFR-3.

Proangiogenic therapies are also important in various diseases (e.g.ischemic heart disease, bleeding etc.), and include VEGF-basedtherapeutics. Activation of VEGFR-2 is proangiogenic (acting onendothelial cells). Activation of VEFGR-1 can cause stimulation ofmigration of inflammatory cells (including, for example, macrophages)and lead to inflammation associated hypervascular permeability.Activation of VEFGR-1 can also promote bone marrow associated tumorniche formation. Thus, VEGF based therapeutic selective for VEGFR-2activation would be desirable in this case. In addition, cell specifictargeting, e.g. to endothelial cells, would be desirable.

In some embodiments, the modified signaling agent has reduced affinityand/or activity (e.g. antagonistic) for VEGFR-2 and/or has substantiallyreduced or ablated affinity and/or activity for VEGFR-1. When targetedto tumor vasculature endothelial cells via a targeting moiety that bindsto a tumor endothelial cell marker (e.g. PSMA and others), suchconstruct inhibits VEGFR-2 activation specifically on suchmarker-positive cells, while not activating VEGFR-1 en route and ontarget cells (if activity ablated), thus eliminating induction ofinflammatory responses, for example. This would provide a more selectiveand safe anti-angiogenic therapy for many tumor types as compared toVEGF-A neutralizing therapies.

In some embodiments, the modified signaling agent has reduced affinityand/or activity (e.g. agonistic) for VEGFR-2 and/or has substantiallyreduced or ablated affinity and/or activity for VEGFR-1. Throughtargeting to vascular endothelial cells, such construct, in someembodiments, promotes angiogenesis without causing VEGFR-1 associatedinduction of inflammatory responses. Hence, such a construct would havetargeted proangiogenic effects with substantially reduced risk of sideeffects caused by systemic activation of VEGFR-2 as well as VEGR-1.

In an illustrative embodiment, the modified signaling agent is VEGF₁₆₅,which has the amino acid sequence:

VEGF 165 (wild type) (SEQ ID NO: 338)APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKAR QLELNERTCRCDKPRR

In another illustrative embodiment, the modified signaling agent isVEGF_(165b), which has the amino acid sequence:

VEGF 165b (wild type) (SEQ ID NO: 339)APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKAR QLELNERTCRSLTRKD

In these embodiments, the modified signaling agent has a mutation atamino acid 183 (e.g., a substitution mutation at 183, e.g., 183K, 183R,or 183H). Without wishing to be bound by theory, it is believed thatsuch mutations may result in reduced receptor binding affinity. See, forexample, U.S. Pat. No. 9,078,860, the entire contents of which arehereby incorporated by reference.

In an embodiment, the modified signaling agent is TNF-α. TNF is apleiotropic cytokine with many diverse functions, including regulationof cell growth, differentiation, apoptosis, tumorigenesis, viralreplication, autoimmunity, immune cell functions and trafficking,inflammation, and septic shock. It binds to two distinct membranereceptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 exhibits avery broad expression pattern whereas TNFR2 is expressed preferentiallyon certain populations of lymphocytes, Tregs, endothelial cells, certainneurons, microglia, cardiac myocytes and mesenchymal stem cells. Verydistinct biological pathways are activated in response to receptoractivation, although there is also some overlap. As a general rule,without wishing to be bound by theory, TNFR1 signaling is associatedwith induction of apoptosis (cell death) and TNFR2 signaling isassociated with activation of cell survival signals (e.g. activation ofNFkB pathway). Administration of TNF is systemically toxic, and this islargely due to TNFR1 engagement. However, it should be noted thatactivation of TNFR2 is also associated with a broad range of activitiesand, as with TNFR1, in the context of developing TNF based therapeutics,control over TNF targeting and activity is important.

In some embodiments, the modified signaling agent has reduced affinityand/or activity for TNFR1 and/or TNFR2. In some embodiments, themodified signaling agent has substantially reduced or ablated affinityand/or activity for TNFR1 and/or TNFR2. TNFR1 is expressed in mosttissues, and is involved in cell death signaling while, by contrast,TNFR2 is involved in cell survival signaling. Accordingly, inembodiments directed to methods of treating cancer, the modifiedsignaling agent has reduced affinity and/or activity for TNFR1 and/orsubstantially reduced or ablated affinity and/or activity for TNFR2. Inthese embodiments, the chimeric proteins may be targeted to a cell forwhich apoptosis is desired, e.g. a tumor cell or a tumor vasculatureendothelial cell. In embodiments directed to methods of promoting cellsurvival, for example, in neurogenesis for the treatment ofneurodegenerative disorders, the modified signaling agent has reducedaffinity and/or activity for TNFR2 and/or substantially reduced orablated affinity and/or activity for TNFR1. Stated another way, thepresent chimeric proteins, in some embodiments, comprise modified TNF-αagent that allows of favoring either death or survival signals.

In some embodiments, the chimeric protein has a modified TNF havingreduced affinity and/or activity for TNFR1 and/or substantially reducedor ablated affinity and/or activity for TNFR2. Such a chimera, in someembodiments, is a more potent inducer of apoptosis as compared to a wildtype TNF and/or a chimera bearing only mutation(s) causing reducedaffinity and/or activity for TNFR1. Such a chimera, in some embodiments,finds use in inducing tumor cell death or a tumor vasculatureendothelial cell death (e.g. in the treatment of cancers). Also, in someembodiments, these chimeras avoid or reduce activation of T_(reg) cellsvia TNFR2, for example, thus further supporting TNFR1-mediated antitumoractivity in vivo.

In some embodiments, the chimeric protein has a modified TNF havingreduced affinity and/or activity for TNFR2 and/or substantially reducedor ablated affinity and/or activity for TNFR1. Such a chimera, in someembodiments, is a more potent activator of cell survival in some celltypes, which may be a specific therapeutic objective in various diseasesettings, including without limitation, stimulation of neurogenesis. Inaddition, such a TNFR2-favoring chimeras also are useful in thetreatment of autoimmune diseases (e.g. Crohn's, diabetes, MS, colitisetc. and many others described herein). In some embodiments, the chimerais targeted to auto-reactive T cells.

In some embodiments, the chimera promotes T_(reg) cell activation andindirect suppression of cytotoxic T cells.

In some embodiments, the chimera causes the death of auto-reactive Tcells, e.g. by activation of TNFR2 and/or avoidance of TNFR1 (e.g. amodified TNF having reduced affinity and/or activity for TNFR2 and/orsubstantially reduced or ablated affinity and/or activity for TNFR1).Without wishing to be bound by theory these auto-reactive T cells, havetheir apoptosis/survival signals altered e.g. by NFkB pathwayactivity/signaling alterations. In some embodiments, the chimera causesthe death of autoreactive T cells having lesions or modifications in theNFkB pathway, which underlie an imbalance of their cell death(apoptosis)/survival signaling properties and, optionally, alteredsusceptibility to certain death-inducing signals (e.g., TNFR2activation).

In some embodiments, a TNFR2 based chimera has additional therapeuticapplications in diseases, including various autoimmune diseases, heartdisease, de-myelinating and neurodegenerative disorders, and infectiousdisease, among others.

In an embodiment, the wild type TNF-α has the amino acid sequence of:

TNF-α (SEQ ID NO: 340) VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQ VYFGIIAL

In such embodiments, the modified TNF-α agent has mutations at one ormore amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146and 147 which produces a modified TNF-α with reduced receptor bindingaffinity. See, for example, U.S. Pat. No. 7,993,636, the entire contentsof which are hereby incorporated by reference.

In some embodiments, the modified human TNF-α moiety has mutations atone or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87,V91, 197, T105, P106, A109, P113, Y115, E127, N137, D143, and A145, asdescribed, for example, in WO/2015/007903, the entire contents of whichis hereby incorporated by reference (numbering according to the humanTNF sequence, Genbank accession number BAG70306, version BAG70306.1 GI:197692685). In some embodiments, the modified human TNF-α moiety hassubstitution mutations selected from R32G, N34G, Q67G, H73G, L75G, L75A,L75S, T77A, S86G, Y87Q, Y87L, Y87A, Y87F, V91G, V91A, 197A, 197Q, 197S,T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G andA145T. In an embodiment, the human TNF-α moiety has a mutation selectedfrom Y87Q, Y87L, Y87A, and Y87F. In another embodiment, the human TNF-αmoiety has a mutation selected from 197A, 197Q, and 197S. In a furtherembodiment, the human TNF-α moiety has a mutation selected from Y115Aand Y115G.

In some embodiments, the modified TNF-α agent has one or more mutationsselected from N39Y, S147Y, and Y87H, as described in WO2008/124086, theentire contents of which is hereby incorporated by reference.

In some embodiments, the modified human TNF-α moiety has mutations thatprovide receptor selectivity as described in PCT/IB2016/001668, theentire contents of which are hereby incorporated by reference. In someembodiments, the mutations to TNF are TNF-R1 selective. In someembodiments, the mutations to TNF which are TNF-R1 selective are at oneor more of positions R32, S86, and E146. In some embodiments, themutations to TNF which are TNF-R1 selective are one or more of R32W,S86T, and E146K. In some embodiments, the mutations to TNF which areTNF-R1 selective are one or more of R32W, R32W/S86T, R32W/E146K andE146K. In some embodiments, the mutations to TNF are TNF-R2 selective.In some embodiments, the mutations to TNF which are TNF-R2 selective areat one or more of positions A145, E146, and S147. In some embodiments,the mutations to TNF which are TNF-R2 selective are one or more ofA145T, A145R, E146D, and S147D. In some embodiments, the mutations toTNF which are TNF-R2 selective are one or more of A145R, A145T/S147D,and A145T/E146D/S 147D.

In an embodiment, the modified signaling agent is TNF-β. TNF-β can forma homotrimer or a heterotrimer with LT-β (LT-α1β2). In some embodiments,the modified signaling agent has substantially reduced or ablatedaffinity and/or activity for TNFR1 and/or TNFR2 and/or herpes virusentry mediator (HEVM) and/or LT-β6R.

In an embodiment, the wild type TNF-β has the amino acid sequence of:

TNF-beta (SEQ ID NO: 341)LPGVGLTPSAAQTARQHPKMHLAHSNLKPAAHLIGDPSKQNSLLWRANTDRAFLQDGFSLSNNSLLVPTSGIYFVYSQWFSGKAYSPKATSSPLYLAHEVQLFSSQYPFHVPLLSSQKMVYPGLQEPWLHSMYHGAAFQLTQGDQLSTH TDGIPHLVLSPSTVFFGAFAL

In such embodiments, the modified TNF-β agent may comprise mutations atone or more amino acids at positions 106-113, which produce a modifiedTNF-β with reduced receptor binding affinity to TNFR2. In an embodiment,the modified signaling agent has one or more substitution mutations atamino acid positions 106-113. In illustrative embodiments, thesubstitution mutations are selected from Q107E, Q107D, S106E, S106D,Q107R, Q107N, Q107E/S106E, Q107E/S106D, Q107D/S106E, and Q107D/S106D. Inanother embodiment, the modified signaling agent has an insertion ofabout 1 to about 3 amino acids at positions 106-113.

In some embodiments, the modified agent is a TNF family member (e.g.TNF-alpha, TNF-beta) which can be a single chain trimeric version asdescribed in WO 2015/007903, the entire contents of which areincorporated by reference.

In some embodiments, the modified agent is a TNF family member (e.g.TNF-alpha, TNF-beta) which has reduced affinity and/or activity, i.e.antagonistic activity (e.g. natural antagonistic activity orantagonistic activity that is the result of one or more mutations, see,e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) at TNFR1. In these embodiments, the modifiedagent is a TNF family member (e.g. TNF-alpha, TNF-beta) which also,optionally, has substantially reduced or ablated affinity and/oractivity for TNFR2. In some embodiments, the modified agent is a TNFfamily member (e.g. TNF-alpha, TNF-beta) which has reduced affinityand/or activity, i.e. antagonistic activity (e.g. natural antagonisticactivity or antagonistic activity that is the result of one or moremutations, see, e.g., WO 2015/007520, the entire contents of which arehereby incorporated by reference) at TNFR2. In these embodiments, themodified agent is a TNF family member (e.g. TNF-alpha, TNF-beta) whichalso, optionally, has substantially reduced or ablated affinity and/oractivity for TNFR1. The constructs of such embodiments find use in, forexample, methods of dampening TNF response in a cell specific manner. Insome embodiments, the antagonistic TNF family member (e.g. TNF-alpha,TNF-beta) is a single chain trimeric version as described in WO2015/007903.

In an embodiment, the modified signaling agent is TRAIL. In someembodiments, the modified TRAIL agent has reduced affinity and/oractivity for DR4 (TRAIL-RI) and/or DR5 (TRAIL-RII) and/or DcR1 and/orDcR2. In some embodiments, the modified TRAIL agent has substantiallyreduced or ablated affinity and/or activity for DR4 (TRAIL-RI) and/orDR5 (TRAIL-RII) and/or DcR1 and/or DcR2.

In an embodiment, the wild type TRAIL has the amino acid sequence of:

TRAIL (SEQ ID NO: 342)MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG

In such embodiments, the modified TRAIL agent may comprise a mutation atamino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209,T214-1220,K224-A226, W231, E236-L239, E249-K251, T261-H264 and H270-E271(Numbering based on the human sequence, Genbank accession numberNP_003801, version 10 NP_003801.1, GI: 4507593; see above).

In an embodiment, the modified signaling agent is TGFα. In suchembodiments, the modified TGFα agent has reduced affinity and/oractivity for the epidermal growth factor receptor (EGFR). In someembodiments, the modified TGFα agent has substantially reduced orablated affinity and/or activity for the epidermal growth factorreceptor (EGFR).

In an embodiment, the modified signaling agent is TGFβ. In suchembodiments, the modified signaling agent has reduced affinity and/oractivity for TGFBR1 and/or TGFBR2. In some embodiments, the modifiedsignaling agent has substantially reduced or ablated affinity and/oractivity for TGFBR1 and/or TGFBR2. In some embodiments, the modifiedsignaling agent optionally has reduced or substantially reduced orablated affinity and/or activity for TGFBR3 which, without wishing to bebound by theory, may act as a reservoir of ligand for TGF-betareceptors. In some embodiments, the TGFβ may favor TGFBR1 over TGFBR2 orTGFBR2 over TGFBR1. Similarly, LAP, without wishing to be bound bytheory, may act as a reservoir of ligand for TGF-beta receptors. In someembodiments, the modified signaling agent has reduced affinity and/oractivity for TGFBR1 and/or TGFBR2 and/or substantially reduced orablated affinity and/or activity for Latency Associated Peptide (LAP).In some embodiments, such chimeras find use in Camurati-Engelmanndisease, or other diseases associated with inappropriate TGFβ signaling.

In some embodiments, the modified agent is a TGF family member (e.g.TGFα, TGFβ) which has reduced affinity and/or activity, i.e.antagonistic activity (e.g. natural antagonistic activity orantagonistic activity that is the result of one or more mutations, see,e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) at one or more of TGFBR1, TGFBR2, TGFBR3. Inthese embodiments, the modified agent is a TGF family member (e.g. TGFα,TGFβ) which also, optionally, has substantially reduced or ablatedaffinity and/or activity at one or more of TGFBR1, TGFBR2, TGFBR3.

In some embodiments, the modified agent is a TGF family member (e.g.TGFα, TGFβ) which has reduced affinity and/or activity, i.e.antagonistic activity (e.g. natural antagonistic activity orantagonistic activity that is the result of one or more mutations, see,e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) at TGFBR1 and/or TGFBR2. In theseembodiments, the modified agent is a TGF family member (e.g. TGFα, TGFβ)which also, optionally, has substantially reduced or ablated affinityand/or activity at TGFBR3.

In an embodiment, the modified signaling agent is an interleukin. In anembodiment, the modified signaling agent is IL-1. In an embodiment, themodified signaling agent is IL-1α or IL-1β. In some embodiments, themodified signaling agent has reduced affinity and/or activity for IL-1R1and/or IL-1RAcP. In some embodiments, the modified signaling agent hassubstantially reduced or ablated affinity and/or activity for IL-1R1and/or IL-1RAcP. In some embodiments, the modified signaling agent hasreduced affinity and/or activity for IL-1R2. In some embodiments, themodified signaling agent has substantially reduced or ablated affinityand/or activity for IL-1R2. For instance, in some embodiments, thepresent modified IL-1 agents avoid interaction at IL-1R2 and thereforesubstantially reduce its function as a decoy and/or sink for therapeuticagents.

In an embodiment, the wild type IL-1β has the amino acid sequence of:

IL-1 beta (mature form, wild type) (SEQ ID NO: 343)APVRSLNCTLRDSQQKSLVMSGPYELKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCVLKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNWYISTSQAENMPVFLGGTKGGQDITDFTMQ FVSS

IL1 is a proinflammatory cytokine and an important immune systemregulator. It is a potent activator of CD4 T cell responses, increasesproportion of Th17 cells and expansion of IFNγ and IL-4 producing cells.IL-1 is also a potent regulator of CD8⁺ T cells, enhancingantigen-specific CD8⁺ T cell expansion, differentiation, migration toperiphery and memory. IL-1 receptors comprise IL-1R1 and IL-1R2. Bindingto and signaling through the IL-1R1 constitutes the mechanism wherebyIL-1 mediates many of its biological (and pathological) activities.IL1-R2 can function as a decoy receptor, thereby reducing IL-1availability for interaction and signaling through the IL-1R1.

In some embodiments, the modified IL-1 has reduced affinity and/oractivity (e.g. agonistic activity) for IL-1R1. In some embodiments, themodified IL-1 has substantially reduced or ablated affinity and/oractivity for IL-1R2. In such embodiments, there is restorableIL-1/IL-1R1 signaling and prevention of loss of therapeutic chimeras atIL-R2 and therefore a reduction in dose of IL-1 that is required (e.g.relative to wild type or a chimera bearing only an attenuation mutationfor IL-R1). Such constructs find use in, for example, methods oftreating cancer, including, for example, stimulating the immune systemto mount an anti-cancer response.

In some embodiments, the modified IL-1 has reduced affinity and/oractivity (e.g. antagonistic activity, e.g. natural antagonistic activityor antagonistic activity that is the result of one or more mutations,see, e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) for IL-1R1. In some embodiments, the modifiedIL-1 has substantially reduced or ablated affinity and/or activity forIL-1R2. In such embodiments, there is the IL-1/IL-1R1 signaling is notrestorable and prevention of loss of therapeutic chimeras at IL-R2 andtherefore a reduction in dose of IL-1 that is required (e.g. relative towild type or a chimera bearing only an attenuation mutation for IL-R1).Such constructs find use in, for example, methods of treating autoimmunediseases, including, for example, suppressing the immune system.

In such embodiments, the modified signaling agent has a deletion ofamino acids 52-54 which produces a modified human IL-1β with reducedbinding affinity for type I IL-1R and reduced biological activity. See,for example, WO 1994/000491, the entire contents of which are herebyincorporated by reference. In some embodiments, the modified human IL-1βhas one or more substitution mutations selected from A117G/P118G, R120X,L122A, T125G/L126G, R127G, Q130X, Q131G, K132A, S137G/Q138Y, L145G,H146X, L145A/L147A, Q148X, Q148G/Q150G, Q150G/D151A, M152G, F162A,F162A/Q164E, F166A, Q164E/E167K, N169G/D170G, I172A, V174A, K208E,K209X, K209A/K210A, K219X, E221X, E221 S/N224A, N224S/K225S, E244K,N245Q (where X can be any change in amino acid, e.g., a non-conservativechange), which exhibit reduced binding to IL-1R, as described, forexample, in WO2015/007542 and WO/2015/007536, the entire contents ofwhich is hereby incorporated by reference (numbering base on the humanIL-1 β sequence, Genbank accession number NP_000567, versionNP-000567.1, GI: 10835145). In some embodiments, the modified humanIL-1β may have one or more mutations selected from R120A, R120G, Q130A,Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A,K209D, K219S, K219Q, E221S and E221K. In an embodiment, the modifiedhuman IL-1β comprises the mutations Q131G and Q148G. In an embodiment,the modified human IL-1β comprises the mutations Q148G and K208E. In anembodiment, the modified human IL-1β comprises the mutations R120G andQ131G. In an embodiment, the modified human IL-1β comprises themutations R120G and H146A. In an embodiment, the modified human IL-1βcomprises the mutations R120G and H146N. In an embodiment, the modifiedhuman IL-1β comprises the mutations R120G and H146R. In an embodiment,the modified human IL-1β comprises the mutations R120G and H146E. In anembodiment, the modified human IL-1β comprises the mutations R120G andH146G. In an embodiment, the modified human IL-1β comprises themutations R120G and K208E. In an embodiment, the modified human IL-1βcomprises the mutations R120G, F162A, and Q164E.

In an embodiment, the modified signaling agent is IL-2. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for IL-2Rα and/or IL-2Rβ and/or IL-α2Rγ. In some embodiments,the modified signaling agent has reduced affinity and/or activity forIL-α2Rβ and/or IL-α2Rγ. In some embodiments, the modified signalingagent has substantially reduced or ablated affinity and/or activity forIL-α2Rα. Such embodiments may be relevant for treatment of cancer, forinstance when the modified IL-2 is agonistic at IL-α2Rβ and/or IL-α2Rγ.For instance, the present constructs may favor attenuated activation ofCD8⁺ T cells (which can provide an anti-tumor effect), which have IL2receptors β and γ and disfavor T_(regs) (which can provide an immunesuppressive, pro-tumor effect), which have IL2 receptors α, β, and γ.Further, in some embodiments, the preferences for IL-α2Rβ and/or IL-α2Rγover IL-α2Rα avoid IL-2 side effects such as pulmonary edema. Also,IL-2-based chimeras are useful for the treatment of autoimmune diseases,for instance when the modified IL-2 is antagonistic (e.g. naturalantagonistic activity or antagonistic activity that is the result of oneor more mutations, see, e.g., WO 2015/007520, the entire contents ofwhich are hereby incorporated by reference) at IL-α2Rβ and/or IL-α2Rγ.For instance, the present constructs may favor attenuated suppression ofCD8⁺ T cells (and therefore dampen the immune response), which have IL2receptors β and γ and disfavor T_(regs) which have IL2 receptors α, β,and γ. Alternatively, in some embodiments, the chimeras bearing IL-2favor the activation of T_(regs), and therefore immune suppression, andactivation of disfavor of CD8⁺ T cells. For instance, these constructsfind use in the treatment of diseases or diseases that would benefitfrom immune suppression, e.g. autoimmune disorders.

In some embodiments, the chimeric protein has targeting moieties asdescribed herein directed to CD8⁺ T cells as well as a modified IL-2agent having reduced affinity and/or activity for IL-α2Rβ and/or IL-α2Rγand/or substantially reduced or ablated affinity and/or activity forIL-α2Rα. In some embodiments, these constructs provide targeted CD8⁺ Tcell activity and are generally inactive (or have substantially reducedactivity) towards T_(reg) cells. In some embodiments, such constructshave enhanced immune stimulatory effect compared to wild type IL-2(e.g., without wishing to be bound by theory, by not stimulating Tregs),whilst eliminating or reducing the systemic toxicity associated withIL-2.

In an embodiment, the wild type IL-2 has the amino acid sequence of:

IL-2 (mature form, wild type) (SEQ ID NO: 344)APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

In such embodiments, the modified IL-2 agent has one or more mutationsat amino acids L72 (L72G, L72A, L725, L72T, L72Q, L72E, L72N, L72D,L72R, or L72K), F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D,F42R, or F42K) and Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D,Y45R or Y45K). Without wishing to be bound by theory, it is believedthat these modified IL-2 agents have reduced affinity for thehigh-affinity IL-2 receptor and preserves affinity to theintermediate-affinity IL-2 receptor, as compared to the wild-type IL-2.See, for example, US Patent Publication No. 2012/0244112, the entirecontents of which are hereby incorporated by reference.

In an embodiment, the modified signaling agent is IL-3. In someembodiments, the modified signaling agent has reduced affinity and/oractivity for the IL-3 receptor, which is a heterodimer with a uniquealpha chain paired with the common beta (beta c or CD131) subunit. Insome embodiments, the modified signaling agent has substantially reducedor ablated affinity and/or activity for the IL-3 receptor, which is aheterodimer with a unique alpha chain paired with the common beta (betac or CD131) subunit.

In an embodiment, the modified signaling agent is IL-4. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for type 1 and/or type 2 IL-4 receptors. In such an embodiment,the modified signaling agent has substantially reduced or ablatedaffinity and/or activity for type 1 and/or type 2 IL-4 receptors. Type 1IL-4 receptors are composed of the IL-4Rα subunit with a common γ chainand specifically bind IL-4. Type 2 IL-4 receptors include an IL-4Rαsubunit bound to a different subunit known as IL-13Rα1. In someembodiments, the modified signaling agent has substantially reduced orablated affinity and/or activity the type 2 IL-4 receptors.

In an embodiment, the wild type IL-4 has the amino acid sequence of:

IL-4 (mature form, wild type) (SEQ ID NO: 345)HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS

In such embodiments, the modified IL-4 agent has one or more mutationsat amino acids R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K,R121N, R121P, R121T, R121W), E122 (E122F), Y124 (Y124A, Y124Q, Y124R,Y124S, Y124T) and S125 (S125A). Without wishing to be bound by theory,it is believed that these modified IL-4 agents maintain the activitymediated by the type I receptor, but significantly reduces thebiological activity mediated by the other receptors. See, for example,U.S. Pat. No. 6,433,157, the entire contents of which are herebyincorporated by reference.

In an embodiment, the modified signaling agent is IL-6. IL-6 signalsthrough a cell-surface type I cytokine receptor complex including theligand-binding IL-6R chain (CD126), and the signal-transducing componentgp130. IL-6 may also bind to a soluble form of IL-6R (sIL-6R), which isthe extracellular portion of IL-6R. The sIL-6R/IL-6 complex may beinvolved in neurites outgrowth and survival of neurons and, hence, maybe important in nerve regeneration through remyelination. Accordingly,in some embodiments, the modified signaling agent has reduced affinityand/or activity for IL-6R/gp130 and/or sIL-6R. In some embodiments, themodified signaling agent has substantially reduced or ablated affinityand/or activity for IL-6R/gp130 and/or sIL-6R.

In an embodiment, the wild type IL-6 has the amino acid sequence of IL-6(mature form, wild type) (SEQ ID NO: 346):

APVPPGEDSKDVAAPHRQPLTSSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMAEKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQMSTKVLIQFLQKKAKNLDAITTPDPTTNASLTTKLQAQNQWLQDMTTHLILRSFKEFLQSSLRALRQM

In such embodiments, the modified signaling agent has one or moremutations at amino acids 58, 160, 163, 171 or 177. Without wishing to bebound by theory, it is believed that these modified IL-6 agents exhibitreduced binding affinity to IL-6Ralpha and reduced biological activity.See, for example, WO 97/10338, the entire contents of which are herebyincorporated by reference.

In an embodiment, the modified signaling agent is IL-10. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for IL-10 receptor-1 and IL-10 receptor-2. In some embodiments,the modified signaling agent has substantially reduced or ablatedaffinity and/or activity for IL-10 receptor-1 and IL-10 receptor-2

In an embodiment, the modified signaling agent is IL-11. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for IL-11Rα and/or IL-11Rβ and/or gp130. In such an embodiment,the modified signaling agent has substantially reduced or ablatedaffinity and/or activity for IL-11Rα and/or IL-11Rβ and/or gp130.

In an embodiment, the modified signaling agent is IL-12. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for IL-12Rβ1 and/or IL-12Rβ2. In such an embodiment, themodified signaling agent has substantially reduced or ablated affinityand/or activity for IL-12Rβ1 and/or IL-12Rβ2.

In an embodiment, the modified signaling agent is IL-13. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for the IL-4 receptor (IL-4Rα) and IL-13Rα1. In someembodiments, the modified signaling agent has substantially reduced orablated affinity and/or activity for IL-4 receptor (IL-4Rα) or IL-13Rα1.

In an embodiment, the wild type IL-13 has the amino acid sequence ofIL-13 (mature form, wild type) (SEQ ID NO: 347):

SPGPVPPSTALRELIEELVNITQNQKAPLCNGSMVWSINLTAGMYCAALESLINVSGCSAIEKTQRMLSGFCPHKVSAGQFSSLHVRDTKIEVAQFVKDL LLHLKKLFREGRFN

In such embodiments, the modified IL-13 agent has one or more mutationsat amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108,109, 112, 113 and 114. Without wishing to be bound by theory, it isbelieved that these modified IL-13 agents exhibit reduced biologicalactivity. See, for example, WO 2002/018422, the entire contents of whichare hereby incorporated by reference.

In an embodiment, the modified signaling agent is IL-18. In someembodiments, the modified signaling agent has reduced affinity and/oractivity for IL-18Rα and/or IL-18Rβ. In some embodiments, the modifiedsignaling agent has substantially reduced or ablated affinity and/oractivity for IL-18Rα and/or IL-18Rβ. In some embodiments, the modifiedsignaling agent has substantially reduced or ablated affinity and/oractivity for IL-18Rα type II, which is an isoform of IL-18Rα that lacksthe TIR domain required for signaling.

In an embodiment, the wild type IL-18 has the amino acid sequence ofIL-18 (wild type) (SEQ ID NO: 348):

MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNEDL

In such embodiments, the modified IL-18 agent may comprise one or moremutations in amino acids or amino acid regions selected from Y37-K44,R49-Q54, D59-R63, E67-C74, R80, M87-A97, N 127-K129, Q139-M149,K165-K171, R183 and Q190-N191, as described in WO/2015/007542, theentire contents of which are hereby incorporated by reference (numberingbased on the human IL-18 sequence, Genbank accession number AAV38697,version AAV38697.1, GI: 54696650).

In an embodiment, the modified signaling agent is IL-33. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for the ST-2 receptor and IL-1RAcP. In some embodiments, themodified signaling agent has substantially reduced or ablated affinityand/or activity for the ST-2 receptor and IL-1RAcP.

In an embodiment, the wild type IL-33 has the amino acid sequence of:

(SEQ ID NO: 349) MKPKMKYSTNKISTAKWKNTASKALCFKLGKSQQKAKEVCPMYFMKLRSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFAFGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEIYVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDFWLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVFIGVKDNHLALI KVDSSENLCTENILFKLSET

In such embodiments, the modified IL-33 agent may comprise one or moremutations in amino acids or amino acid regions selected from 1113-Y122,5127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227 and T260-E269, asdescribed in WO/2015/007542, the entire contents of which are herebyincorporated by reference (numbering based on the human sequence,Genbank accession number NP_254274, version NP_254274.1, GI:15559209).

In an embodiment, the modified signaling agent is epidermal growthfactor (EGF). EGF is a member of a family of potent growth factors.Members include EGF, HB-EGF, and others such as TGFalpha, amphiregulin,neuregulins, epiregulin, betacellulin. EGF family receptors include EGFR(ErbB1), ErbB2, ErbB3 and ErbB4. These may function as homodimericand/or heterodimeric receptor subtypes. The different EGF family membersexhibit differential selectivity for the various receptor subtypes. Forexample, EGF associates with ErbB1/ErbB1, ErbB1/ErbB2, ErbB4/ErbB2 andsome other heterodimeric subtypes. HB-EGF has a similar pattern,although it also associates with ErbB4/4. Modulation of EGF (EGF-like)growth factor signaling, positively or negatively, is of considerabletherapeutic interest. For example, inhibition of EGFRs signaling is ofinterest in the treatment of various cancers where EGFR signalingconstitutes a major growth promoting signal. Alternatively, stimulationof EGFRs signaling is of therapeutic interest in, for example, promotingwound healing (acute and chronic), oral mucositis (a major side-effectof various cancer therapies, including, without limitation radiationtherapy).

In some embodiments, the modified signaling agent has reduced affinityand/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4. Such embodimentsfind use, for example, in methods of treating wounds. In someembodiments, the modified signaling agent binds to one or more ErbB1,ErbB2, ErbB3, and ErbB4 and antagonizes the activity of the receptor. Insuch embodiments, the modified signaling agent has reduced affinityand/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4 which allows forthe activity of the receptor to be antagonized in an attenuated fashion.Such embodiments find use in, for example, treatments of cancer. In anembodiment, the modified signaling agent has reduced affinity and/oractivity for ErbB1. ErbB1 is the therapeutic target of kinaseinhibitors-most have side effects because they are not very selective(e.g., gefitinib, erlotinib, afatinib, brigatinib and icotinib). In someembodiments, attenuated antagonistic ErbB1 signaling is more on-targetand has less side effects than other agents targeting receptors for EGF.

In some embodiments, the modified signaling agent has reduced affinityand/or activity (e.g. antagonistic e.g. natural antagonistic activity orantagonistic activity that is the result of one or more mutations, see,e.g., WO 2015/007520, the entire contents of which are herebyincorporated by reference) for ErbB1 and/or substantially reduced orablated affinity and/or activity for ErbB4 or other subtypes it mayinteract with. Through specific targeting via the targeting moiety,cell-selective suppression (antagonism e.g. natural antagonisticactivity or antagonistic activity that is the result of one or moremutations, see, e.g., WO 2015/007520, the entire contents of which arehereby incorporated by reference) of ErbB1/ErbB1 receptor activationwould be achieved—while not engaging other receptor subtypes potentiallyassociated with inhibition-associated side effects. Hence, in contrastto EGFR kinase inhibitors, which inhibit EGFR activity in all cell typesin the body, such a construct would provide a cell-selective (e.g.,tumor cell with activated EGFR signaling due to amplification ofreceptor, overexpression etc.) anti-EGFR (ErbB1) drug effect withreduced side effects.

In some embodiments, the modified signaling agent has reduced affinityand/or activity (e.g. agonistic) for ErbB4 and/or other subtypes it mayinteract with. Through targeting to specific target cells through thetargeting moiety, a selective activation of ErbB1 signaling is achieved(e.g. epithelial cells). Such a construct finds use, in someembodiments, in the treatment of wounds (promoting would healing) withreduced side effects, especially for treatment of chronic conditions andapplication other than topical application of a therapeutic (e.g.systemic wound healing).

In an embodiment, the modified signaling agent is insulin or insulinanalogs. In some embodiments, the modified insulin or insulin analog hasreduced affinity and/or activity for the insulin receptor and/or IGF1 orIGF2 receptor. In some embodiments, the modified insulin or insulinanalog has substantially reduced or ablated affinity and/or activity forthe insulin receptor and/or IGF1 or IGF2 receptor. Attenuated responseat the insulin receptor allows for the control of diabetes, obesity,metabolic disorders and the like while directing away from IGF1 or IGF2receptor avoids pro-cancer effects.

In an embodiment, the modified signaling agent is insulin-like growthfactor-I or insulin-like growth factor-II (IGF-1 or IGF-2). In anembodiment, the modified signaling agent is IGF-1. In such anembodiment, the modified signaling agent has reduced affinity and/oractivity for the insulin receptor and/or IGF1 receptor. In anembodiment, the modified signaling agent may bind to the IGF1 receptorand antagonize the activity of the receptor. In such an embodiment, themodified signaling agent has reduced affinity and/or activity for IGF1receptor which allows for the activity of the receptor to be antagonizedin an attenuated fashion. In some embodiments, the modified signalingagent has substantially reduced or ablated affinity and/or activity forthe insulin receptor and/or IGF1 receptor. In some embodiments, themodified signaling agent has reduced affinity and/or activity for IGF2receptor which allows for the activity of the receptor to be antagonizedin an attenuated fashion. In an embodiment, the modified signaling agenthas substantially reduced or ablated affinity and/or activity for theinsulin receptor and accordingly does not interfere with insulinsignaling. In various embodiments, this applies to cancer treatment. Invarious embodiments, the present agents may prevent IR isoform A fromcausing resistance to cancer treatments.

In an embodiment, the modified signaling agent is EPO. In variousembodiments, the modified EPO agent has reduced affinity and/or activityfor the EPO receptor (EPOR) receptor and/or the ephrin receptor (EphR)relative to wild type EPO or other EPO based agents described herein. Insome embodiments, the modified EPO agent has substantially reduced orablated affinity and/or activity for the EPO receptor (EPOR) receptorand/or the Eph receptor (EphR). Illustrative EPO receptors include, butare not limited to, an EPOR homodimer or an EPOR/CD131 heterodimer. Alsoincluded as an EPO receptor is beta-common receptor (βcR). IllustrativeEph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3,EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3,EPHB4, EPHB5, and EPHB6. In some embodiments, the modified EPO proteincomprises one or more mutations that cause the EPO protein to havereduced affinity for receptors that comprise one or more different EPOreceptors or Eph receptors (e.g. heterodimer, heterotrimers, etc.,including by way of non-limitation: EPOR-EPHB4, EPOR-βcR-EPOR). Alsoprovided are the receptors of EP Patent Publication No. 2492355 theentire contents of which are hereby incorporated by reference, includingby way of non-limitation, NEPORs.

In an embodiment, the human EPO has the amino acid sequence of (thesignal peptide is underlined):

(SEQ ID NO: 350) MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGK LKLYTGEACRTGDR

In an embodiment, the human EPO protein is the mature form of EPO (withthe signal peptide being cleaved off) which is a glycoprotein of 166amino acid residues having the sequence of:

(SEQ ID NO: 351) APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

The structure of the human EPO protein is predicted to comprisefour-helix bundles including helices A, B, C, and D. In variousembodiments, the modified EPO protein comprises one or more mutationslocated in four regions of the EPO protein which are important forbioactivity, i.e., amino acid residues 10-20, 44-51, 96-108, and142-156. In some embodiments, the one or more mutations are located atresidues 11-15, 44-51, 100-108, and 147-151. These residues arelocalized to helix A (Val11, Arg14, and Tyr15), helix C (Ser100, Arg103,Ser104, and Leu108), helix D (Asn147, Arg150, Gly151, and Leu155), andthe NB connecting loop (residues 42-51). In some embodiments, themodified EPO protein comprises mutations in residues between amino acids41-52 and amino acids 147, 150, 151, and 155. Without wishing to bebound by theory, it is believed that mutations of these residues havesubstantial effects on both receptor binding and in vitro biologicalactivity. In some embodiments, the modified EPO protein comprisesmutations at residues 11, 14, 15, 100, 103, 104, and 108. Withoutwishing to be bound by theory, it is believed that mutations of theseresidues have modest effects on receptor binding activity and muchgreater effects on in vitro biological activity. Illustrativesubstitutions include, but are not limited to, one or more of Val11Ser,Arg14Ala, Arg14Gln, Tyr15Ile, Pro42Asn, Thr44Ile, Lys45Asp, Val46Ala,Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104Ile, Ser104Ala,Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155Ala.

In some embodiments, the modified EPO protein comprises mutations thateffect bioactivity and not binding, e.g. those listed in Eliot, et al.Mapping of the Active Site of Recombinant Human Erythropoietin Jan. 15,1997; Blood: 89 (2), the entire contents of which are herebyincorporated by reference.

In some embodiments, the modified EPO protein comprises one or moremutations involving surface residues of the EPO protein which areinvolved in receptor contact. Without wishing to be bound by theory, itis believed that mutations of these surface residues are less likely toaffect protein folding thereby retaining some biological activity.Illustrative surface residues that may be mutated include, but are notlimited to, residues 147 and 150. In illustrative embodiments, themutations are substitutions including, one or more of N147A, N147K,R150A and R150E.

In some embodiments, the modified EPO protein comprises one or moremutations at residues N59, E62, L67, and L70, and one or more mutationsthat affect disulfide bond formation. Without wishing to be bound bytheory, it is believed that these mutations affect folding and/or arepredicted be in buried positions and thus affects biological activityindirectly.

In an embodiment, the modified EPO protein comprises a K20E substitutionwhich significantly reduces receptor binding. See Elliott, et al.,(1997) Blood, 89:493-502, the entire contents of which are herebyincorporated by reference.

Additional EPO mutations that may be incorporated into the chimeric EPOprotein of the invention are disclosed in, for example, Elliott, et al.,(1997) Blood, 89:493-502, the entire contents of which are herebyincorporated by reference and Taylor et al., (2010) PEDS, 23(4):251-260, the entire contents of which are hereby incorporated byreference.

In some embodiments, the chimeric protein of the invention comprises amodified IL-1 (e.g., IL-1α and/or IL-1β) as the signaling agent. In suchembodiments, the chimeric protein has effects on (and one targetingmoiety targets) T cells such as T helper cells (e.g. for co-stimulationand activation) and/or B cells (e.g. for maturation, proliferation),and/or NK cells (e.g. for activation) and/or macrophages (e.g. foractivation).

In some embodiments, the chimeric protein of the invention comprises amodified IL-2 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for growth and differentiation) and/or B cells (e.g. for growth) and/orNK cells (e.g. for growth) and/or macrophages.

In some embodiments, the chimeric protein of the invention comprises amodified IL-3 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) hematopoieticstem cells (e.g., for growth, differentiation and proliferation) and/ormyeloid progenitor cells (e.g. for differentiation and proliferation)and/or mast cells (e.g. for growth).

In some embodiments, the chimeric protein of the invention comprises amodified IL-4 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for growth, proliferation, and survival) and/or B cells (e.g., foractivation, growth, differentiation, and proliferation) and/or mastcells (e.g. for growth) and/or macrophages (e.g., for inhibition ofactivation).

In some embodiments, the chimeric protein of the invention comprises amodified IL-5 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) eosinophils(e.g. for growth and differentiation) and/or B cells (e.g. fordifferentiation and production).

In some embodiments, the chimeric protein of the invention comprises amodified IL-6 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) B cells (e.g.,for growth and differentiation) and/or plasma cells (e.g. for antibodysecretion) and/or hematopoietic stem cells (e.g. for differentiation)and/or T cells (e.g. for growth and differentiation).

In some embodiments, the chimeric protein of the invention comprises amodified IL-7 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for growth and differentiation and survival) and/or B cells (e.g. forgrowth and differentiation and survival) and/or NK cells (e.g. forgrowth and differentiation and survival).

In some embodiments, the chimeric protein of the invention comprises amodified IL-8 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) neutrophilsand/or basophils and/or B cells and/or T cells and/or NK cells.

In some embodiments, the chimeric protein of the invention comprises amodified IL-9 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells and/orB cells (e.g. activation) and/or mast cells (e.g. stimulation).

In some embodiments, the chimeric protein of the invention comprises amodified IL-10 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) macrophages(e.g. for inhibition) and/or B cells (e.g. for activation) and/or mastcells (e.g. for co-stimulation of growth) and/or Th₁ cells (e.g. forinhibition) and/or Th₂ cells (e.g. for stimulation).

In some embodiments, the chimeric protein of the invention comprises amodified IL-12 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for differentiation) and/or NK cells (e.g. for activation).

In some embodiments, the chimeric protein of the invention comprises amodified IL-13 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.,Th₁ and Th₂ cells; for example, for inhibition of Th₁ cells) and/or Bcells (e.g. for growth and differentiation) and/or macrophages (e.g.,for inhibition).

In some embodiments, the chimeric protein of the invention comprises amodified IL-14 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) B cells (e.g.,for growth and proliferation).

In some embodiments, the chimeric protein of the invention comprises amodified IL-15 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for growth) and/or B cells and or NK cells (e.g. for growth).

In some embodiments, the chimeric protein of the invention comprises amodified IL-16 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.,CD4+ T cells; for example, for survival).

In some embodiments, the chimeric protein of the invention comprises amodified IL-18 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) Th₁ cells(e.g., for induction) and/or NK cells (e.g., for induction of IFNγproduction).

In some embodiments, the chimeric protein of the invention comprises amodified IL-21 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for activation and proliferation and/or differentiation) and/or B cells(e.g. for proliferation and differentiation) and/or NK cells and/ordendritic cells.

In some embodiments, the chimeric protein of the invention comprises amodified IL-27 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells and/orB cells.

In some embodiments, the chimeric protein of the invention comprises amodified IL-32 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) monocytesand/or macrophages.

In some embodiments, the chimeric protein of the invention comprises amodified IL-33 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.,T helper cells).

In some embodiments, the chimeric protein of the invention comprises amodified IL-35 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.,T helper cells).

In some embodiments, the chimeric protein of the invention comprises amodified IL-36 as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells and/ordendritic cells.

In some embodiments, the chimeric protein of the invention comprises amodified TNF-α as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) macrophages(e.g., for activation).

In some embodiments, the chimeric protein of the invention comprises amodified TNF-β as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for killing) and/or B cells (e.g., for inhibition) and/or macrophages(e.g., for activation) and/or neutrophils (e.g. for activation).

In some embodiments, the chimeric protein of the invention comprises amodified CD40L as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) B cells (e.g.for activation).

In some embodiments, the chimeric protein of the invention comprises amodified CD27L as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for proliferation).

In some embodiments, the chimeric protein of the invention comprises amodified CD30L as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for proliferation) and/or B cells (e.g. for proliferation).

In some embodiments, the chimeric protein of the invention comprises amodified 4-1BBL as the signaling agent. In such embodiments, thechimeric protein has effects on (and one targeting moiety targets) Tcells (e.g. for co-stimulation) and/or B cells (e.g. forco-stimulation).

In some embodiments, the chimeric protein of the invention comprises amodified TGF-β as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) B cells (e.g.for inhibition of growth) and/or macrophages (e.g. for inhibition ofactivation) and/or neutrophils (e.g. for activation).

In some embodiments, the chimeric protein of the invention comprises amodified IFN-γ as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) T cells (e.g.for inhibition of Th₂ growth) and/or B cells (e.g. for differentiation)and/or NK cells (e.g. for activation) and/or macrophages (e.g. foractivation).

In some embodiments, the chimeric protein of the invention comprises amodified GM-CSF as the signaling agent. In such embodiments, thechimeric protein has effects on (and one targeting moiety targets) Tcells (e.g. for inhibition of growth) and/or B cells (e.g. fordifferentiation) and/or macrophages (e.g. for activation anddifferentiation) and hematopoietic stem cells (e.g. for differentiation)and/or myeloid progenitor cells (e.g. for growth and differentiation).

In some embodiments, the chimeric protein of the invention comprises amodified G-CSF as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) neutrophils(e.g. for development and differentiation).

In some embodiments, the chimeric protein of the invention comprises amodified EPO as the signaling agent. In such embodiments, the chimericprotein has effects on (and one targeting moiety targets) hematopoieticprogenitor and/or precursor cells and/or erythroid progenitor and/orprecursor cells (e.g., BFU-E (burst forming unit-erythroid) and/or CFU-E(colony-forming units-erythroid)).

By way of non-limiting example, in one embodiment, the present chimericprotein has (i) a targeting moiety directed against a T cell, forexample, mediated by targeting to one of CD8, CD4, and CD3 and (ii) atargeting moiety is directed against a tumor cell (e.g., mediated bytargeting to, without limitation, a checkpoint inhibitor, e.g., withoutlimitation, PD-L1 or PD-L2), along with any of the modified (e.g.mutant) signaling agents described herein, for example, modified (e.g.mutant) signaling agent that may alter the activity of a T cell, forexample, interferon alpha, including without limitation, one or more offollowing mutations F64A, N65A, T69A, L80A, Y85A, Y89A, R120E,R120E/K121E, K133A, R144A, M148A, R149A, and L153A. In an embodiment,the present chimeric protein has a targeting moiety directed against CD8on T cells, a second targeting moiety directed against a checkpointinhibitor, e.g., without limitation, PD-L1 or PD-L2, on tumor cells, anda modified (e.g. mutant) interferon agent as described herein.

By way of non-limiting example, in one embodiment, the present chimericprotein has (i) a targeting moiety directed against a B cell, forexample, mediated by targeting to CD20 and (ii) a targeting moiety isdirected against a tumor cell (e.g., mediated by targeting to, withoutlimitation, a checkpoint inhibitor, e.g., without limitation, PD-L1 orPD-L2), along with any of the modified (e.g. mutant) signaling agentsdescribed herein, for example, modified (e.g. mutant) signaling agentthat may alter the activity of a B cell, for example, interferon alpha,including without limitation, one or more of the following mutationsF64A, N65A, T69A, L80A, Y85A, Y89A, R120E, R120E/K121E, K133A, R144A,M148A, R149A, and L153A.

By way of non-limiting example, in one embodiment, the present chimericprotein has (i) a targeting moiety directed against a dendritic cell,for example, mediated by targeting to one of CLEC9A, XCR1, and RANK and(ii) a targeting moiety is directed against a tumor cell (e.g., mediatedby targeting to, without limitation, a checkpoint inhibitor, e.g.,without limitation, PD-L1 or PD-L2), along with any of the modified(e.g. mutant) signaling agents described herein, for example, modified(e.g. mutant) signaling agent that may alter the activity of a dendriticcell, for example, interferon alpha, including without limitation, oneor more of the following mutations F64A, N65A, T69A, L80A, Y85A, Y89A,R120E, R120E/K121E, K133A, R144A, M148A, R149A, and L153A. In anembodiment, the present chimeric protein has a targeting moiety directedagainst CLEC9A on dendritic cells, a second targeting moiety directedagainst a checkpoint inhibitor, e.g., without limitation, PD-L1 orPD-L2, on tumor cells, and a modified (e.g. mutant) interferon agent asdescribed herein.

By way of non-limiting example, in one embodiment, the present chimericprotein has (i) a targeting moiety directed against a macrophage, forexample, mediated by targeting to SIRP1a and (ii) a targeting moiety isdirected against a tumor cell (e.g., mediated by targeting to, withoutlimitation, a checkpoint inhibitor, e.g., without limitation, PD-L1 orPD-L2), along with any of the modified (e.g. mutant) signaling agentsdescribed herein, for example, modified (e.g. mutant) signaling agentthat may alter the activity of a macrophage, for example, interferonalpha, including without limitation, one or more of the followingmutations F64A, N65A, T69A, L80A, Y85A, Y89A, R120E, R120E/K121E, K133A,R144A, M148A, R149A, and L153A.

By way of non-limiting example, in one embodiment, the present chimericprotein has (i) a targeting moiety directed against a NK cell, forexample, mediated by targeting to TIGIT or KIR1 and (ii) a targetingmoiety is directed against a tumor cell (e.g., mediated by targeting to,without limitation, a checkpoint inhibitor, e.g., without limitation,PD-L1 or PD-L2), along with any of the modified (e.g. mutant) signalingagents described herein, for example, modified (e.g. mutant) signalingagent that may alter the activity of a NK cell, for example, interferonalpha, including without limitation, one or more of the followingmutations F64A, N65A, T69A, L80A, Y85A, Y89A, R120E, R120E/K121E, K133A,R144A, M148A, R149A, and L153A.

In various embodiments, the signaling agent is a toxin or toxic enzyme.In some embodiments, the toxin or toxic enzyme is derived from plantsand bacteria. Illustrative toxins or toxic enzymes include, but are notlimited to, the diphtheria toxin, Pseudomonas toxin, anthrax toxin,ribosome-inactivating proteins (RIPs) such as ricin and saporin,modeccin, abrin, gelonin, and poke weed antiviral protein. Additionaltoxins include those disclosed in Mathew et al., (2009) Cancer Sci100(8): 1359-65, the entire disclosures are hereby incorporated byreference. In such embodiments, the chimeric proteins of the inventionmay be utilized to induce cell death in cell-type specific manner. Insuch embodiments, the toxin may be modified, e.g. mutated, to reduceaffinity and/or activity of the toxin for an attenuated effect, asdescribed with other signaling agents herein.

Linkers

In some embodiments, the present chimeric protein optionally comprisesone or more linkers. In some embodiments, the present chimeric proteincomprises a linker connecting the targeting moiety and the signalingagent. In some embodiments, the present chimeric protein comprises alinker within the signaling agent (e.g. in the case of single chain TNF,which can comprise two linkers to yield a trimer).

In some embodiments vectors encoding the present chimeric proteinslinked as a single nucleotide sequence to any of the linkers describedherein are provided and may be used to prepare such chimeric proteins.

In some embodiments, the linker length allows for efficient binding of atargeting moiety and the signaling agent to their receptors. Forinstance, in some embodiments, the linker length allows for efficientbinding of one of the targeting moieties and the signaling agent toreceptors on the same cell as well as the efficient binding of the othertargeting moiety to another cell. Illustrative pairs of cells areprovided elsewhere herein.

In some embodiments the linker length is at least equal to the minimumdistance between the binding sites of one of the targeting moieties andthe signaling agent to receptors on the same cell. In some embodimentsthe linker length is at least twice, or three times, or four times, orfive times, or ten times, or twenty times, or 25 times, or 50 times, orone hundred times, or more the minimum distance between the bindingsites of one of the targeting moieties and the signaling agent toreceptors on the same cell.

As described herein, the linker length allows for efficient binding ofone of the targeting moieties and the signaling agent to receptors onthe same cell, the binding being sequential, e.g. targetingmoiety/receptor binding preceding signaling agent/receptor binding.

In some embodiments, there are two linkers in a single chimera, eachconnecting the signaling agent to a targeting moiety. In variousembodiments, the linkers have lengths that allow for the formation of asite that has a disease cell and an effector cell without sterichindrance that would prevent modulation of the either cell.

The invention contemplates the use of a variety of linker sequences. Invarious embodiments, the linker may be derived from naturally-occurringmulti-domain proteins or are empirical linkers as described, forexample, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen etal., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contentsof which are hereby incorporated by reference. In some embodiments, thelinker may be designed using linker designing databases and computerprograms such as those described in Chen et al., (2013), Adv Drug DelivRev. 65(10):1357-1369 and Crasto et al., (2000), Protein Eng.13(5):309-312, the entire contents of which are hereby incorporated byreference. In various embodiments, the linker may be functional. Forexample, without limitation, the linker may function to improve thefolding and/or stability, improve the expression, improve thepharmacokinetics, and/or improve the bioactivity of the present chimericprotein.

In some embodiments, the linker is a polypeptide. In some embodiments,the linker is less than about 100 amino acids long. For example, thelinker may be less than about 100, about 95, about 90, about 85, about80, about 75, about 70, about 65, about 60, about 55, about 50, about45, about 40, about 35, about 30, about 25, about 20, about 19, about18, about 17, about 16, about 15, about 14, about 13, about 12, about11, about 10, about 9, about 8, about 7, about 6, about 5, about 4,about 3, or about 2 amino acids long. In some embodiments, the linker isa polypeptide. In some embodiments, the linker is greater than about 100amino acids long. For example, the linker may be greater than about 100,about 95, about 90, about 85, about 80, about 75, about 70, about 65,about 60, about 55, about 50, about 45, about 40, about 35, about 30,about 25, about 20, about 19, about 18, about 17, about 16, about 15,about 14, about 13, about 12, about 11, about 10, about 9, about 8,about 7, about 6, about 5, about 4, about 3, or about 2 amino acidslong. In some embodiments, the linker is flexible. In anotherembodiment, the linker is rigid.

In some embodiments, a linker connects the two targeting moieties toeach other and this linker has a short length and a linker connects atargeting moiety and a signaling agent this linker is longer than thelinker connecting the two targeting moieties. For example, thedifference in amino acid length between the linker connecting the twotargeting moieties and the linker connecting a targeting moiety and asignaling agent may be about 100, about 95, about 90, about 85, about80, about 75, about 70, about 65, about 60, about 55, about 50, about45, about 40, about 35, about 30, about 25, about 20, about 19, about18, about 17, about 16, about 15, about 14, about 13, about 12, about11, about 10, about 9, about 8, about 7, about 6, about 5, about 4,about 3, or about 2 amino acids.

In various embodiments, the linker is substantially comprised of glycineand serine residues (e.g. about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90%, or about 95%, orabout 97% glycines and serines). For example, in some embodiments, thelinker is (Gly₄Ser)_(n), where n is from about 1 to about 8, e.g. 1, 2,3, 4, 5, 6, 7, or 8. In an embodiment, the linker sequence isGGSGGSGGGGSGGGGS (SEQ ID NO: 352). Additional illustrative linkersinclude, but are not limited to, linkers having the sequence LE, GGGGS(SEQ ID NO: 353), (GGGGS)_(n) (n=1-4) (SEQ ID NO: 354), (Gly)₈ (SEQ IDNO: 355), (Gly)₆ (SEQ ID NO: 356), (EAAAK)_(n) (n=1-3) (SEQ ID NO: 357),A(EAAAK)_(n)A (n=2-5) (SEQ ID NO: 358), AEAAAKEAAAKA (SEQ ID NO: 359),A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 360), PAPAP (SEQ ID NO: 361),KESGSVSSEQLAQFRSLD (SEQ ID NO: 362), EGKSSGSGSESKST (SEQ ID NO: 363),GSAGSAAGSGEF (SEQ ID NO: 364), and (XP)_(n), with X designating anyamino acid, e.g., Ala, Lys, or Glu. In various embodiments, the linkeris GGS.

In some embodiments, the linker is a hinge region of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linkeris a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE,inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4, and IgA1 andIgA2)). The hinge region, found in IgG, IgA, IgD, and IgE classantibodies, acts as a flexible spacer, allowing the Fab portion to movefreely in space. In contrast to the constant regions, the hinge domainsare structurally diverse, varying in both sequence and length amongimmunoglobulin classes and subclasses. For example, the length andflexibility of the hinge region varies among the IgG subclasses. Thehinge region of IgG1 encompasses amino acids 216-231 and, because it isfreely flexible, the Fab fragments can rotate about their axes ofsymmetry and move within a sphere centered at the first of twointer-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1,with 12 amino acid residues and four disulfide bridges. The hinge regionof IgG2 lacks a glycine residue, is relatively short, and contains arigid poly-proline double helix, stabilized by extra inter-heavy chaindisulfide bridges. These properties restrict the flexibility of the IgG2molecule. IgG3 differs from the other subclasses by its unique extendedhinge region (about four times as long as the IgG1 hinge), containing 62amino acids (including 21 prolines and 11 cysteines), forming aninflexible poly-proline double helix. In IgG3, the Fab fragments arerelatively far away from the Fc fragment, giving the molecule a greaterflexibility. The elongated hinge in IgG3 is also responsible for itshigher molecular weight compared to the other subclasses. The hingeregion of IgG4 is shorter than that of IgG1 and its flexibility isintermediate between that of IgG1 and IgG2. The flexibility of the hingeregions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of CH1 to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). Id. The core hinge region of wild-type human IgG1 contains thesequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In various embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g. IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In various embodiments, the linker of thepresent invention comprises one or more glycosylation sites. In variousembodiments, the linker is a hinge-CH2-CH3 domain of a human IgG4antibody.

If desired, the present chimeric protein can be linked to an antibody Fcregion, comprising one or both of C_(H)2 and C_(H)3 domains, andoptionally a hinge region. For example, vectors encoding the presentchimeric proteins linked as a single nucleotide sequence to an Fc regioncan be used to prepare such polypeptides.

In some embodiments, the linker is a synthetic linker such as PEG.

In various embodiments, the linker may be functional. For example,without limitation, the linker may function to improve the foldingand/or stability, improve the expression, improve the pharmacokinetics,and/or improve the bioactivity of the present chimeric protein. Inanother example, the linker may function to target the chimeric proteinto a particular cell type or location.

Production of Chimeric Proteins

Methods for producing the chimeric proteins of the invention aredescribed herein. For example, DNA sequences encoding the chimericproteins of the invention (e.g., DNA sequences encoding the modifiedsignaling agent and the targeting moiety and the linker) can bechemically synthesized using methods known in the art. Synthetic DNAsequences can be ligated to other appropriate nucleotide sequences,including, e.g., expression control sequences, to produce geneexpression constructs encoding the desired chimeric proteins.Accordingly, in various embodiments, the present invention provides forisolated nucleic acids comprising a nucleotide sequence encoding thechimeric protein of the invention.

Nucleic acids encoding the chimeric protein of the invention can beincorporated (ligated) into expression vectors, which can be introducedinto host cells through transfection, transformation, or transductiontechniques. For example, nucleic acids encoding the chimeric protein ofthe invention can be introduced into host cells by retroviraltransduction. Illustrative host cells are E. coli cells, Chinese hamsterovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells.Transformed host cells can be grown under conditions that permit thehost cells to express the genes that encode the chimeric protein of theinvention. Accordingly, in various embodiments, the present inventionprovides expression vectors comprising nucleic acids that encode thechimeric protein of the invention. In various embodiments, the presentinvention additional provides host cells comprising such expressionvectors.

Specific expression and purification conditions will vary depending uponthe expression system employed. For example, if a gene is to beexpressed in E. coli, it is first cloned into an expression vector bypositioning the engineered gene downstream from a suitable bacterialpromoter, e.g., Trp or Tac, and a prokaryotic signal sequence. Inanother example, if the engineered gene is to be expressed in eukaryotichost cells, e.g., CHO cells, it is first inserted into an expressionvector containing for example, a suitable eukaryotic promoter, asecretion signal, enhancers, and various introns. The gene construct canbe introduced into the host cells using transfection, transformation, ortransduction techniques.

The chimeric protein of the invention can be produced by growing a hostcell transfected with an expression vector encoding the chimeric proteinunder conditions that permit expression of the protein. Followingexpression, the protein can be harvested and purified using techniqueswell known in the art, e.g., affinity tags such asglutathione-S-transferase (GST) and histidine tags or by chromatography.

Accordingly, in various embodiments, the present invention provides fora nucleic acid encoding a chimeric protein of the present invention. Invarious embodiments, the present invention provides for a host cellcomprising a nucleic acid encoding a chimeric protein of the presentinvention.

Pharmaceutically Acceptable Salts and Excipients

The chimeric proteins described herein can possess a sufficiently basicfunctional group, which can react with an inorganic or organic acid, ora carboxyl group, which can react with an inorganic or organic base, toform a pharmaceutically acceptable salt. A pharmaceutically acceptableacid addition salt is formed from a pharmaceutically acceptable acid, asis well known in the art. Such salts include the pharmaceuticallyacceptable salts listed in, for example, Journal of PharmaceuticalScience, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts;Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.),Verlag, Zurich (Switzerland) 2002, which are hereby incorporated byreference in their entirety.

Pharmaceutically acceptable salts include, by way of non-limitingexample, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide,nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate,phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate,hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycollate,heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate,mesylate, nicotinate, phthalate, teraphthalate, propiolate, propionate,phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate,chlorobenzenesulfonate, ethylsulfonate, 2-hydroxyethylsulfonate,methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,naphthalene-1,5-sulfonate, xylenesulfonate, and tartarate salts.

The term “pharmaceutically acceptable salt” also refers to a salt of thecompositions of the present invention having an acidic functional group,such as a carboxylic acid functional group, and a base. Suitable basesinclude, but are not limited to, hydroxides of alkali metals such assodium, potassium, and lithium; hydroxides of alkaline earth metal suchas calcium and magnesium; hydroxides of other metals, such as aluminumand zinc; ammonia, and organic amines, such as unsubstituted orhydroxy-substituted mono-, di-, or tri-alkylamines, dicyclohexylamine;tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine;triethylamine; mono-, bis-, or tris-(2-OH-lower alkylamines), such asmono-; bis-, or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine,or tris-(hydroxymethyl)methylamine, N,N-di-lower alkyl-N-(hydroxyl-loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like.

In some embodiments, the compositions described herein are in the formof a pharmaceutically acceptable salt.

Pharmaceutical Compositions and Formulations

In various embodiments, the present invention pertains to pharmaceuticalcompositions comprising the chimeric proteins described herein and apharmaceutically acceptable carrier or excipient. Any pharmaceuticalcompositions described herein can be administered to a subject as acomponent of a composition that comprises a pharmaceutically acceptablecarrier or vehicle. Such compositions can optionally comprise a suitableamount of a pharmaceutically acceptable excipient so as to provide theform for proper administration.

In various embodiments, pharmaceutical excipients can be liquids, suchas water and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. The pharmaceutical excipients can be, for example,saline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea and the like. In addition, auxiliary, stabilizing,thickening, lubricating, and coloring agents can be used. In oneembodiment, the pharmaceutically acceptable excipients are sterile whenadministered to a subject. Water is a useful excipient when any agentdescribed herein is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidexcipients, specifically for injectable solutions. Suitablepharmaceutical excipients also include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents. Other examples of suitablepharmaceutical excipients are described in Remington's PharmaceuticalSciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed. 1995),incorporated herein by reference.

The present invention includes the described pharmaceutical compositions(and/or additional therapeutic agents) in various formulations. Anyinventive pharmaceutical composition (and/or additional therapeuticagents) described herein can take the form of solutions, suspensions,emulsion, drops, tablets, pills, pellets, capsules, capsules containingliquids, gelatin capsules, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, lyophilizedpowder, frozen suspension, dessicated powder, or any other form suitablefor use. In one embodiment, the composition is in the form of a capsule.In another embodiment, the composition is in the form of a tablet. Inyet another embodiment, the pharmaceutical composition is formulated inthe form of a soft-gel capsule. In a further embodiment, thepharmaceutical composition is formulated in the form of a gelatincapsule. In yet another embodiment, the pharmaceutical composition isformulated as a liquid.

Where necessary, the inventive pharmaceutical compositions (and/oradditional agents) can also include a solubilizing agent. Also, theagents can be delivered with a suitable vehicle or delivery device asknown in the art. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.

The formulations comprising the inventive pharmaceutical compositions(and/or additional agents) of the present invention may conveniently bepresented in unit dosage forms and may be prepared by any of the methodswell known in the art of pharmacy. Such methods generally include thestep of bringing the therapeutic agents into association with a carrier,which constitutes one or more accessory ingredients. Typically, theformulations are prepared by uniformly and intimately bringing thetherapeutic agent into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct into dosage forms of the desired formulation (e.g., wet or drygranulation, powder blends, etc., followed by tableting usingconventional methods known in the art).

In various embodiments, any pharmaceutical compositions (and/oradditional agents) described herein is formulated in accordance withroutine procedures as a composition adapted for a mode of administrationdescribed herein.

Routes of administration include, for example: oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically. Administration canbe local or systemic. In some embodiments, the administering is effectedorally. In another embodiment, the administration is by parenteralinjection. The mode of administration can be left to the discretion ofthe practitioner, and depends in-part upon the site of the medicalcondition. In most instances, administration results in the release ofany agent described herein into the bloodstream.

In one embodiment, the chimeric protein described herein is formulatedin accordance with routine procedures as a composition adapted for oraladministration. Compositions for oral delivery can be in the form oftablets, lozenges, aqueous or oily suspensions, granules, powders,emulsions, capsules, syrups, or elixirs, for example. Orallyadministered compositions can comprise one or more agents, for example,sweetening agents such as fructose, aspartame or saccharin; flavoringagents such as peppermint, oil of wintergreen, or cherry; coloringagents; and preserving agents, to provide a pharmaceutically palatablepreparation. Moreover, where in tablet or pill form, the compositionscan be coated to delay disintegration and absorption in thegastrointestinal tract thereby providing a sustained action over anextended period of time. Selectively permeable membranes surrounding anosmotically active driving any chimeric proteins described herein arealso suitable for orally administered compositions. In these latterplatforms, fluid from the environment surrounding the capsule is imbibedby the driving compound, which swells to displace the agent or agentcomposition through an aperture. These delivery platforms can provide anessentially zero order delivery profile as opposed to the spikedprofiles of immediate release formulations. A time-delay material suchas glycerol monostearate or glycerol stearate can also be useful. Oralcompositions can include standard excipients such as mannitol, lactose,starch, magnesium stearate, sodium saccharin, cellulose, and magnesiumcarbonate. In one embodiment, the excipients are of pharmaceuticalgrade. Suspensions, in addition to the active compounds, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth,etc., and mixtures thereof.

Dosage forms suitable for parenteral administration (e.g. intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g. lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art. Formulation components suitable forparenteral administration include a sterile diluent such as water forinjection, saline solution, fixed oils, polyethylene glycols, glycerine,propylene glycol or other synthetic solvents; antibacterial agents suchas benzyl alcohol or methyl paraben; antioxidants such as ascorbic acidor sodium bisulfite; chelating agents such as EDTA; buffers such asacetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose.

For intravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) orphosphate buffered saline (PBS). The carrier should be stable under theconditions of manufacture and storage, and should be preserved againstmicroorganisms. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyetheylene glycol), and suitablemixtures thereof.

The compositions provided herein, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e.,“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Any inventive pharmaceutical compositions (and/or additional agents)described herein can be administered by controlled-release orsustained-release means or by delivery devices that are well known tothose of ordinary skill in the art. Examples include, but are notlimited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899;3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767;5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of whichis incorporated herein by reference in its entirety. Such dosage formscan be useful for providing controlled- or sustained-release of one ormore active ingredients using, for example, hydropropyl cellulose,hydropropylmethyl cellulose, polyvinylpyrrolidone, other polymermatrices, gels, permeable membranes, osmotic systems, multilayercoatings, microparticles, liposomes, microspheres, or a combinationthereof to provide the desired release profile in varying proportions.Suitable controlled- or sustained-release formulations known to thoseskilled in the art, including those described herein, can be readilyselected for use with the active ingredients of the agents describedherein. The invention thus provides single unit dosage forms suitablefor oral administration such as, but not limited to, tablets, capsules,gelcaps, and caplets that are adapted for controlled- orsustained-release.

Controlled- or sustained-release of an active ingredient can bestimulated by various conditions, including but not limited to, changesin pH, changes in temperature, stimulation by an appropriate wavelengthof light, concentration or availability of enzymes, concentration oravailability of water, or other physiological conditions or compounds.

In another embodiment, a controlled-release system can be placed inproximity of the target area to be treated, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer,1990, Science 249:1527-1533) may be used.

Pharmaceutical formulations preferably are sterile. Sterilization can beaccomplished, for example, by filtration through sterile filtrationmembranes. Where the composition is lyophilized, filter sterilizationcan be conducted prior to or following lyophilization andreconstitution.

Administration and Dosage

It will be appreciated that the actual dose of the chimeric protein tobe administered according to the present invention will vary accordingto the particular dosage form, and the mode of administration. Manyfactors that may modify the action of the chimeric protein (e.g., bodyweight, gender, diet, time of administration, route of administration,rate of excretion, condition of the subject, drug combinations, geneticdisposition and reaction sensitivities) can be taken into account bythose skilled in the art. Administration can be carried out continuouslyor in one or more discrete doses within the maximum tolerated dose.Optimal administration rates for a given set of conditions can beascertained by those skilled in the art using conventional dosageadministration tests.

In some embodiments, a suitable dosage of the chimeric protein is in arange of about 0.01 mg/kg to about 10 g/kg of body weight of thesubject, about 0.01 mg/kg to about 1 g/kg of body weight of the subject,about 0.01 mg/kg to about 100 mg/kg of body weight of the subject, about0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example,about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg,about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg,about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg,about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg,about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg,about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, about 100mg/kg body weight, about 1 g/kg of body weight, about 10 g/kg of bodyweight, inclusive of all values and ranges therebetween.

Individual doses of the chimeric protein can be administered in unitdosage forms (e.g., tablets or capsules) containing, for example, fromabout 0.01 mg to about 100 g, from about 0.01 mg to about 75 g, fromabout 0.01 mg to about 50 g, from about 0.01 mg to about 25 g, about0.01 mg to about 10 g, about 0.01 mg to about 7.5 g, about 0.01 mg toabout 5 g, about 0.01 mg to about 2.5 g, about 0.01 mg to about 1 g,about 0.01 mg to about 100 mg, from about 0.1 mg to about 100 mg, fromabout 0.1 mg to about 90 mg, from about 0.1 mg to about 80 mg, fromabout 0.1 mg to about 70 mg, from about 0.1 mg to about 60 mg, fromabout 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg activeingredient, from about 0.1 mg to about 30 mg, from about 0.1 mg to about20 mg, from about 0.1 mg to about 10 mg, from about 0.1 mg to about 5mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 1 mg perunit dosage form, or from about 5 mg to about 80 mg per unit dosageform. For example, a unit dosage form can be about 0.01 mg, about 0.02mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg,about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg about 10 mg,about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg,about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g,about 5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g,inclusive of all values and ranges therebetween.

In one embodiment, the chimeric protein is administered at an amount offrom about 0.01 mg to about 100 g daily, from about 0.01 mg to about 75g daily, from about 0.01 mg to about 50 g daily, from about 0.01 mg toabout 25 g daily, from about 0.01 mg to about 10 g daily, from about0.01 mg to about 7.5 g daily, from about 0.01 mg to about 5 g daily,from about 0.01 mg to about 2.5 g daily, from about 0.01 mg to about 1 gdaily, from about 0.01 mg to about 100 mg daily, from about 0.1 mg toabout 100 mg daily, from about 0.1 mg to about 95 mg daily, from about0.1 mg to about 90 mg daily, from about 0.1 mg to about 85 mg daily,from about 0.1 mg to about 80 mg daily, from about 0.1 mg to about 75 mgdaily, from about 0.1 mg to about 70 mg daily, from about 0.1 mg toabout 65 mg daily, from about 0.1 mg to about 60 mg daily, from about0.1 mg to about 55 mg daily, from about 0.1 mg to about 50 mg daily,from about 0.1 mg to about 45 mg daily, from about 0.1 mg to about 40 mgdaily, from about 0.1 mg to about 35 mg daily, from about 0.1 mg toabout 30 mg daily, from about 0.1 mg to about 25 mg daily, from about0.1 mg to about 20 mg daily, from about 0.1 mg to about 15 mg daily,from about 0.1 mg to about 10 mg daily, from about 0.1 mg to about 5 mgdaily, from about 0.1 mg to about 3 mg daily, from about 0.1 mg to about1 mg daily, or from about 5 mg to about 80 mg daily. In variousembodiments, the chimeric protein is administered at a daily dose ofabout 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8mg, about 9 mg about 10 mg, about 15 mg, about 20 mg, about 25 mg, about30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg,about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500mg, about 1 g, about 2.5 g, about 5 g, about 7.5 g, about 10 g, about 25g, about 50 g, about 75 g, about 100 g, inclusive of all values andranges therebetween.

In accordance with certain embodiments of the invention, thepharmaceutical composition comprising the chimeric protein may beadministered, for example, more than once daily (e.g., about two times,about three times, about four times, about five times, about six times,about seven times, about eight times, about nine times, or about tentimes daily), about once per day, about every other day, about everythird day, about once a week, about once every two weeks, about onceevery month, about once every two months, about once every three months,about once every six months, or about once every year.

Combination Therapy and Additional Therapeutic Agents

In various embodiments, the pharmaceutical composition of the presentinvention is co-administered in conjunction with additional therapeuticagent(s). Co-administration can be simultaneous or sequential.

In one embodiment, the additional therapeutic agent and the chimericprotein of the present invention are administered to a subjectsimultaneously. The term “simultaneously” as used herein, means that theadditional therapeutic agent and the chimeric protein are administeredwith a time separation of no more than about 60 minutes, such as no morethan about 30 minutes, no more than about 20 minutes, no more than about10 minutes, no more than about 5 minutes, or no more than about 1minute. Administration of the additional therapeutic agent and thechimeric protein can be by simultaneous administration of a singleformulation (e.g., a formulation comprising the additional therapeuticagent and the chimeric protein) or of separate formulations (e.g., afirst formulation including the additional therapeutic agent and asecond formulation including the chimeric protein).

Co-administration does not require the therapeutic agents to beadministered simultaneously, if the timing of their administration issuch that the pharmacological activities of the additional therapeuticagent and the chimeric protein overlap in time, thereby exerting acombined therapeutic effect. For example, the additional therapeuticagent and the chimeric protein can be administered sequentially. Theterm “sequentially” as used herein means that the additional therapeuticagent and the chimeric protein are administered with a time separationof more than about 60 minutes. For example, the time between thesequential administration of the additional therapeutic agent and thechimeric protein can be more than about 60 minutes, more than about 2hours, more than about 5 hours, more than about 10 hours, more thanabout 1 day, more than about 2 days, more than about 3 days, more thanabout 1 week apart, more than about 2 weeks apart, or more than aboutone month apart. The optimal administration times will depend on therates of metabolism, excretion, and/or the pharmacodynamic activity ofthe additional therapeutic agent and the chimeric protein beingadministered. Either the additional therapeutic agent or the chimericprotein cell may be administered first.

Co-administration also does not require the therapeutic agents to beadministered to the subject by the same route of administration. Rather,each therapeutic agent can be administered by any appropriate route, forexample, parenterally or non-parenterally.

In some embodiments, the chimeric protein described herein actssynergistically when co-administered with another therapeutic agent. Insuch embodiments, the chimeric protein and the additional therapeuticagent may be administered at doses that are lower than the dosesemployed when the agents are used in the context of monotherapy.

In some embodiments, the present invention pertains to chemotherapeuticagents as additional therapeutic agents. For example, withoutlimitation, such combination of the present chimeric proteins andchemotherapeutic agent find use in the treatment of cancers, asdescribed elsewhere herein. Examples of chemotherapeutic agents include,but are not limited to, alkylating agents such as thiotepa and CYTOXANcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), andTAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb); inhibitors of PKC-α, Raf, H-Ras,EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, the methods of treatmentcan further include the use of radiation. In addition, the methods oftreatment can further include the use of photodynamic therapy.

In some embodiments, the present bi-specific chimeras are combined withFMS-like tyrosine kinase 3 ligand (FLT3L), e.g. anti-human Clec9aVHH/anti-human PDL1 VHH/human IFN-R149A bi-specific chimera can becombined with FLT3L.

In some embodiments, inclusive of, without limitation, infectiousdisease applications, the present invention pertains to anti-infectivesas additional therapeutic agents. In some embodiments, theanti-infective is an anti-viral agent including, but not limited to,Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir,Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir,Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet. Insome embodiments, the anti-infective is an anti-bacterial agentincluding, but not limited to, cephalosporin antibiotics (cephalexin,cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole,cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics(cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracyclineantibiotics (tetracycline, minocycline, oxytetracycline, anddoxycycline); penicillin antibiotics (amoxicillin, ampicillin,penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); monobactam antibiotics (aztreonam); and carbapenemantibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).In some embodiments, the anti-infectives include anti-malarial agents(e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline,artemether/lumefantrine, atovaquone/proguanil andsulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin,pyrantel pamoate, and albendazole.

In some embodiments, inclusive, without limitation, of autoimmmuneapplications, the additional therapeutic agent is an immunosuppressiveagent. In some embodiments, the immunosuppressive agent is ananti-inflammatory agent such as a steroidal anti-inflammatory agent or anon-steroidal anti-inflammatory agent (NSAID). Steroids, particularlythe adrenal corticosteroids and their synthetic analogues, are wellknown in the art. Examples of corticosteroids useful in the presentinvention include, without limitation, hydroxyltriamcinolone,alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasonedipropionate, betamethasone benzoate, betamethasone dipropionate,betamethasone valerate, clobetasol valerate, desonide, desoxymethasone,dexamethasone, diflorasone diacetate, diflucortolone valerate,fluadrenolone, fluclorolone acetonide, flumethasone pivalate,fluosinolone acetonide, fluocinonide, flucortine butylester,fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone,halcinonide, hydrocortisone acetate, hydrocortisone butyrate,methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone,flucetonide, fludrocortisone, difluorosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, clocortelone, clescinolone,dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone,fluperolone, fluprednisolone, hydrocortisone, meprednisone,paramethasone, prednisolone, prednisone, beclomethasone dipropionate.(NSAIDS) that may be used in the present invention, include but are notlimited to, salicylic acid, acetyl salicylic acid, methyl salicylate,glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid,ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone,and indomethacin. In some embodiments, the immunosupressive agent may becytostatics such as alkylating agents, antimetabolites (e.g.,azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g.,basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g.,cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF bindingproteins, mycophenolates, and small biological agents (e.g., fingolimod,myriocin). Additional anti-inflammatory agents are described, forexample, in U.S. Pat. No. 4,537,776, the entire contents of which isincorporated by reference herein.

In some embodiments, the present invention pertains to various agentsused for treating obesity as additional therapeutic agents. Illustrativeagents used for treating obesity include, but are not limited to,orlistat (e.g. ALL1, XENICAL), loracaserin (e.g. BELVIQ),phentermine-topiramate (e.g. QSYMIA), sibutramme (e.g. REDUCTIL orMERJDIA), rimonabant (ACOMPLLA), exenatide (e.g. BYETTA), pramlintide(e.g. SYMLIN) phentermine, benzphetamine, diethylpropion,phendimetrazme, bupropion, and metformin. Agents that interfere with thebody's ability to absorb specific nutrients in food are among theadditional agents, e.g. orlistat (e.g. ALU, XENICAL), glucomannan, andguar gum. Agents that suppress apetite are also among the additionalagents, e.g. catecholamines and their derivatives (such as phenteimineand other amphetamine-based drugs), various antidepressants and moodstabilizers (e.g. bupropion and topiramate), anorectics (e.g. dexedrine,digoxin). Agents that increase the body's metabolism are also among theadditional agents.

In some embodiments, additional therapeutic agents may be selected fromamong appetite suppressants, neurotransmitter reuptake inhibitors,dopaminergic agonists, serotonergic agonists, modulators of GABAergicsignaling, anticonvulsants, antidepressants, monoamine oxidaseinhibitors, substance P (NK1) receptor antagonists, melanocortinreceptor agonists and antagonists, lipase inhibitors, inhibitors of fatabsorption, regulators of energy intake or metabolism, cannabinoidreceptor modulators, agents for treating addiction, agents for treatingmetabolic syndrome, peroxisome proliferator-activated receptor (PPAR)modulators; dipcptidyl peptidase 4 (DPP-4) antagonists, agents fortreating cardiovascular disease, agents for treating elevatedtriglyceride levels, agents for treating low HDL, agents for treatinghypercholesterolemia, and agents for treating hypertension. Some agentsfor cardiovascular disease include statins (e.g. lovastatin,atorvastatin, fluvastatin, rosuvastatin, simvastatin and pravastatin)and omega-3 agents (e.g. LOVAZA, EPANQVA, VASCEPA, esterified omega-3'sin general, fish oils, krill oils, algal oils). In some embodiments,additional agents may be selected from among amphetamines,benzodiazepines, sulfonyl ureas, meglitinides, thiazolidinediones,biguanides, beta-blockers, XCE inhibitors, diuretics, nitrates, calciumchannel blockers, phenlermine, sibutramine, iorcaserin, cetilistat,rimonabant, taranabant, topiramate, gabapentin, valproate, vigabatrin,bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide,methylphenidate, varenicline, naltrexone, diethylpropion,phendimetrazine, rcpaglinide, nateglinide, glimepiride, metformin,pioglitazone, rosiglilazone, and sitagliptin.

In some embodiments, the present invention pertains to an agent used fortreating diabetes as additional therapeutic agents. Illustrativeanti-diabetic agents include, but are not limited to, sulfonylurea(e.g., DYMELOR (acetohexamide), DIABINESE (chlorpropamide), ORINASE(tolbutamide), and TOLINASE (tolazamide), GLUCOTROL (glipizide),GLUCOTROL XL (extended release), DIABETA (glyburide), MICRONASE(glyburide), GLYNASE PRESTAB (glyburide), and AMARYL (glimepiride)); aBiguanide (e.g. metformin (GLUCOPHAGE, GLUCOPHAGE XR, RIOMET, FORTAMET,and GLUMETZA)); a thiazolidinedione (e.g. ACTOS (pioglitazone) andAVANDIA (rosiglitazone); an alpha-glucosidase inhibitor (e.g., PRECOSE(acarbose) and GLYSET (miglitol); a Meglitinide (e.g., PRANDIN(repaglinide) and STARLIX (nateglinide)); a Dipeptidyl peptidase IV(DPP-IV) inhibitor (e.g., JANUVIA (sitagliptin), NESINA (alogliptin),ONGLYZA (saxagliptin), and TRADJENTA (linagliptin)); Sodium-glucoseco-transporter 2 (SGLT2) inhibitor (e.g. INVOKANA (canaglifozin)); and acombination pill (e.g. GLUCOVANCE, which combines glyburide (asulfonylurea) and metformin, METAGLIP, which combines glipizide (asulfonylurea) and metformin, and AVANDAMET, which uses both metforminand rosiglitazone (AVANDIA) in one pill, KAZANO (alogliptin andmetformin), OSENI (alogliptin plus pioglitazone), METFORMIN oral, ACTOSoral, BYETTA subcutaneous, JANUVIA oral, WELCHOL oral, JANUMET oral,glipizide oral, glimepiride oral, GLUCOPHAGE oral, LANTUS subcutaneous,glyburide oral, ONGLYZA oral, AMARYI oral, LANTUS SOLOSTAR subcutaneous,BYDUREON subcutaneous, LEVEMIR FLEXPEN subcutaneous, ACTOPLUS MET oral,GLUMETZA oral, TRADJENTA oral, bromocriptine oral, KOMBIGLYZE XR oral,INVOKANA oral, PRANDIN oral, LEVEMIR subcutaneous, PARLODEL oral,pioglitazone oral, NOVOLOG subcutaneous, NOVOLOG FLEXPEN subcutaneous,VICTOZA 2-PAK subcutaneous, HUMALOG subcutaneous, STARLIX oral, FORTAMEToral, GLUCOVANCE oral, GLUCOPHAGE XR oral, NOVOLOG Mix 70-30 FLEXPENsubcutaneous, GLYBURIDE-METFORMIN oral, acarbose oral, SYMLINPEN 60subcutaneous, GLUCOTROI XL oral, NOVOLIN R inj, GLUCOTROL oral, DUETACToral, sitagliptin oral, SYMLINPEN 120 subcutaneous, HUMALOG KWIKPENsubcutaneous, JANUMET XR oral, GLIPIZIDE-METFORMIN oral, CYCLOSET oral,HUMALOG MIX 75-25 subcutaneous, nateglinide oral, HUMALOG Mix 75-25KWIKPEN subcutaneous, HUMULIN 70/30 subcutaneous, PRECOSE oral, APIDRAsubcutaneous, Humulin R inj, Jentadueto oral, Victoza 3-Paksubcutaneous, Novolin 70/30 subcutaneous, NOVOLIN N subcutaneous,insulin detemir subcutaneous, glyburide micronized oral, GLYNASE oral,HUMULIN N subcutaneous, insulin glargine subcutaneous, RIOMET oral,pioglitazone-metformin oral, APIDRA SOLOSTAR subcutaneous, insulinlispro subcutaneous, GLYSET oral, HUMULIN 70/30 Pen subcutaneous,colesevelam oral, sitagliptin-metformin oral, DIABETA oral, insulinregular human inj, HUMULIN N Pen subcutaneous, exenatide subcutaneous,HUMALOG Mix 50-50 KWIKPEN subcutaneous, liraglutide subcutaneous, KAZANOoral, repaglinide oral, chlorpropamide oral, insulin aspartsubcutaneous, NOVOLOG Mix 70-30 subcutaneous, HUMALOG Mix 50-50subcutaneous, saxagliptin oral, ACTOPLUS Met XR oral, miglitol oral, NPHinsulin human recomb subcutaneous, insulin NPH and regular humansubcutaneous, tolazamide oral, mifepristone oral, insulin aspartprotam-insulin aspart subcutaneous, repaglinide-metformin oral,saxagliptin-metformin oral, linagliptin-metformin oral, NESINA oral,OSENI oral, tolbutamide oral, insulin lispro protamine and lisprosubcutaneous, pramlintide subcutaneous, insulin glulisine subcutaneous,pioglitazone-glimepiride oral, PRANDIMET oral, NOVOLOG PenFillsubcutaneous, linagliptin oral, exenatide microspheres subcutaneous,KORLYM oral, alogliptin oral, alogliptin-pioglitazone oral,alogliptin-metformin oral, canagliflozin oral, Lispro (HUMALOG); Aspart(NOVOLOG); Glulisine (APIDRA); Regular (NOVOLIN R or HUMULIN R); NPH(NOVOLIN N or HUMULIN N); Glargine (LANTUS); Detemir (LEVEMIR); HUMULINor NOVOLIN 70/30; and NOVOLOG Mix 70/30 HUMALOG Mix 75/25 or 50/50.

In some embodiments, the present invention relates to combinationtherapy with a blood transfusion. For instance, the present compositionsmay supplement a blood transfusion. In some embodiments, the presentinvention relates to combination therapy with iron supplements.

In some embodiments, the present invention relates to combinationtherapy with one or more EPO-based agents. For example, the presentcompositions may be used as an adjuvant to other EPO-based agents. Insome embodiments, the present compositions are used as a maintenancetherapy to other EPO-based agents. Other EPO-based agents include thefollowing: epoetin alfa, including without limitation, DARBEPOETIN(ARANESP), EPOCEPT (LUPIN PHARMA), NANOKINE (NANOGEN PHARMACEUTICAL),EPOFIT (INTAS PHARMA), EPOGEN (AMGEN), EPOGIN, EPREX, (JANSSEN-CILAG),BINOCRIT (SANDOZ), PROCRIT; epoetin beta, including without limitation,NEORECORMON (HOFFMANN-LA ROCHE), RECORMON, Methoxy polyethyleneglycol-epoetin beta (MIRCERA, ROCHE); epoetin delta, including withoutlimitation, DYNEPO (erythropoiesis stimulating protein, SHIRE PLC);epoetin omega, including without limitation, EPOMAX; epoetin zeta,including without limitation, SILAPO (STADA) and RETACRIT (HOSPIRA) andother EPOs, including without limitation, EPOCEPT (LUPINPHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ERYPRO SAFE (BIOCONLTD.), REPOITIN (SERUM INSTITUTE OF INDIA LIMITED), VINTOR (EMCUREPHARMACEUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTASBIOPHARMACEUTICA), WEPDX (WOCKHARDT BIOTECH), ESPOGEN (LG LIFESCIENCES), RELIPOIETIN (RELIANCE LIFE SCIENCES), SHANPOIETIN (SHANTHABIOTECHNICS LTD), ZYROP (CADILA HEALTHCARE LTD.), EPIAO (RHUEPO)(SHENYANG SUNSHINE PHARMACEUTICAL CO. LTD), CINNAPOIETIN (CINNAGEN).

In some embodiments, the present invention relates to combinationtherapy with one or more immune-modulating agents, for example, withoutlimitation, agents that modulate immune checkpoint. In variousembodiments, the immune-modulating agent targets one or more of PD-1,PD-L1, and PD-L2. In various embodiments, the immune-modulating agent isPD-1 inhibitor. In various embodiments, the immune-modulating agent isan antibody specific for one or more of PD-1, PD-L1, and PD-L2. Forinstance, in some embodiments, the immune-modulating agent is anantibody such as, by way of non-limitation, nivolumab,(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL3280A (ROCHE).In some embodiments, the immune-modulating agent targets one or more ofCD137 or CD137L. In various embodiments, the immune-modulating agent isan antibody specific for one or more of CD137 or CD137L. For instance,in some embodiments, the immune-modulating agent is an antibody such as,by way of non-limitation, urelumab (also known as BMS-663513 andanti-4-1BB antibody). In some embodiments, the present chimeric proteinis combined with urelumab (optionally with one or more of nivolumab,lirilumab, and urelumab) for the treatment of solid tumors and/or B-cellnon-Hodgkins lymphoma and/or head and neck cancer and/or multiplemyeloma. In some embodiments, the immune-modulating agent is an agentthat targets one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2, andPPP2R5A. In various embodiments, the immune-modulating agent is anantibody specific for one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2,and PPP2R5A. For instance, in some embodiments, the immune-modulatingagent is an antibody such as, by way of non-limitation, ipilimumab(MDX-010, MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). In someembodiments, the present chimeric protein is combined with ipilimumab(optionally with bavituximab) for the treatment of one or more ofmelanoma, prostate cancer, and lung cancer. In various embodiments, theimmune-modulating agent targets CD20. In various embodiments, theimmune-modulating agent is an antibody specific CD20. For instance, insome embodiments, the immune-modulating agent is an antibody such as, byway of non-limitation, Ofatumumab (GENMAB), obinutuzumab (GAZYVA),AME-133v (APPLIED MOLECULAR EVOLUTION), Ocrelizumab (GENENTECH), TRU-015(TRUBION/EMERGENT), veltuzumab (IMMU-106).

In some embodiments, the present invention relates to combinationtherapy with one or more chimeric agents described in WO 2013/10779, WO2015/007536, WO 2015/007520, WO 2015/007542, and WO 2015/007903, theentire contents of which are hereby incorporated by reference in theirentireties.

In some embodiments, the chimeric protein described herein, includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the composition such that covalent attachment doesnot prevent the activity of the composition. For example, but not by wayof limitation, derivatives include composition that have been modifiedby, inter alia, glycosylation, lipidation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc.

In still other embodiments, the chimeric protein described hereinfurther comprise a cytotoxic agent, comprising, in illustrativeembodiments, a toxin, a chemotherapeutic agent, a radioisotope, and anagent that causes apoptosis or cell death. Such agents may be conjugatedto a composition described herein.

The chimeric protein described herein may thus be modifiedpost-translationally to add effector moieties such as chemical linkers,detectable moieties such as for example fluorescent dyes, enzymes,substrates, bioluminescent materials, radioactive materials, andchemiluminescent moieties, or functional moieties such as for examplestreptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, andradioactive materials.

Illustrative cytotoxic agents include, but are not limited to,methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine; alkylating agents such as mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C,lomustine (CCNU), 1-methylnitrosourea, cyclothosphamide,mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C,cis-dichlorodiamine platinum (II) (DDP) cisplatin and carboplatin(paraplatin); anthracyclines include daunorubicin (formerly daunomycin),doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin,epirubicin, mitoxantrone and bisantrene; antibiotics includedactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, andanthramycin (AMC); and antimytotic agents such as the vinca alkaloids,vincristine and vinblastine. Other cytotoxic agents include paclitaxel(taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B,gramicidin D, ethidium bromide, emetine, etoposide, tenoposide,colchicin, dihydroxy anthracin dione, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids,mytotane (O,P′-(DDD)), interferons, and mixtures of these cytotoxicagents.

Further cytotoxic agents include, but are not limited to,chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C,actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGFantagonists, EGFR antagonists, platins, taxols, irinotecan,5-fluorouracil, gemcytabine, leucovorine, steroids, cyclophosphamide,melphalan, vinca alkaloids (e.g., vinblastine, vincristine, vindesineand vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy,sex hormone antagonists, selective androgen receptor modulators,selective estrogen receptor modulators, PDGF antagonists, TNFantagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12Rantagonists, Toxin conjugated monoclonal antibodies, tumor antigenspecific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20antibodies, Rituxan, ocrelizumab, ofatumumab, DXL625, HERCEPTIN®, or anycombination thereof. Toxic enzymes from plants and bacteria such asricin, diphtheria toxin and Pseudomonas toxin may be conjugated to thetherapeutic agents (e.g. antibodies) to generatecell-type-specific-killing reagents (Youle, et al., Proc. Nat'l Acad.Sci. USA 77:5483 (1980); Gilliland, et al., Proc. Nat'l Acad. Sci. USA77:4539 (1980); Krolick, et al., Proc. Nat'l Acad. Sci. USA 77:5419(1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described byGoldenberg in U.S. Pat. No. 6,653,104. Embodiments of the invention alsorelate to radioimmunoconjugates where a radionuclide that emits alpha orbeta particles is stably coupled to the chimeric protein, with orwithout the use of a complex-forming agent. Such radionuclides includebeta-emitters such as Phosphorus-32, Scandium-47, Copper-67, Gallium-67,Yttrium-88, Yttrium-90, Iodine-125, Iodine-131, Samarium-153,Lutetium-177, Rhenium-186 or Rhenium-188, and alpha-emitters such asAstatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.

Illustrative detectable moieties further include, but are not limitedto, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase,beta-galactosidase and luciferase. Further illustrative fluorescentmaterials include, but are not limited to, rhodamine, fluorescein,fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine,phycoerythrin and dansyl chloride. Further illustrative chemiluminescentmoieties include, but are not limited to, luminol. Further illustrativebioluminescent materials include, but are not limited to, luciferin andaequorin. Further illustrative radioactive materials include, but arenot limited to, Iodine-125, Carbon-14, Sulfur-35, Tritium andPhosphorus-32.

Methods of Treatment

Methods and compositions described herein have application to treatingvarious diseases and disorders, including, but not limited to cancer,infections, immune disorders, anemia, autoimmune diseases,cardiovascular diseases, wound healing, ischemia-related diseases,neurodegenerative diseases, metabolic diseases and many other diseasesand disorders.

Further, any of the present agents may be for use in the treating, orthe manufacture of a medicament for treating, various diseases anddisorders, including, but not limited to cancer, infections, immunedisorders, inflammatory diseases or conditions, and autoimmune diseases.

In some embodiments, the present invention relates to the treatment of,or a patient having one or more of cancer, heart failure, autoimmunedisease, sickle cell disease, thalassemia, blood loss, transfusionreaction, diabetes, vitamin B12 deficiency, collagen vascular disease,Shwachman syndrome, thrombocytopenic purpura, Celiac disease, endocrinedeficiency state such as hypothyroidism or Addison's disease, autoimmunedisease such as Crohn's Disease, systemic lupus erythematosis,rheumatoid arthritis or juvenile rheumatoid arthritis, ulcerativecolitis immune disorders such as eosinophilic fasciitis,hypoimmunoglobulinemia, or thymoma/thymic carcinoma, graft versus hostdisease, preleukemia, Nonhematologic syndrome (e.g. Down's, Dubowwitz,Seckel), Felty syndrome, hemolytic uremic syndrome, myelodysplasicsyndrome, nocturnal paroxysmal hemoglobinuria, osteomyelofibrosis,pancytopenia, pure red-cell aplasia, Schoenlein-Henoch purpura, malaria,protein starvation, menorrhagia, systemic sclerosis, liver cirrhosis,hypometabolic states, and congestive heart failure.

In some embodiments, the present invention relates to the treatment of,or a patient having cancer. As used herein, cancer refers to anyuncontrolled growth of cells that may interfere with the normalfunctioning of the bodily organs and systems, and includes both primaryand metastatic tumors. Primary tumors or cancers that migrate from theiroriginal location and seed vital organs can eventually lead to the deathof the subject through the functional deterioration of the affectedorgans. A metastasis is a cancer cell or group of cancer cells, distinctfrom the primary tumor location, resulting from the dissemination ofcancer cells from the primary tumor to other parts of the body.Metastases may eventually result in death of a subject. For example,cancers can include benign and malignant cancers, polyps, hyperplasia,as well as dormant tumors or micrometastases.

Illustrative cancers that may be treated include, but are not limitedto, carcinomas, e.g. various subtypes, including, for example,adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, andtransitional cell carcinoma), sarcomas (including, for example, bone andsoft tissue), leukemias (including, for example, acute myeloid, acutelymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cell),lymphomas and myelomas (including, for example, Hodgkin and non-Hodgkinlymphomas, light chain, non-secretory, MGUS, and plasmacytomas), andcentral nervous system cancers (including, for example, brain (e.g.gliomas (e.g. astrocytoma, oligodendroglioma, and ependymoma),meningioma, pituitary adenoma, and neuromas, and spinal cord tumors(e.g. meningiomas and neurofibroma).

Illustrative cancers that may be treated include, but are not limitedto, basal cell carcinoma, biliary tract cancer; bladder cancer; bonecancer; brain and central nervous system cancer; breast cancer; cancerof the peritoneum; cervical cancer; choriocarcinoma; colon and rectumcancer; connective tissue cancer; cancer of the digestive system;endometrial cancer; esophageal cancer; eye cancer; cancer of the headand neck; gastric cancer (including gastrointestinal cancer);glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm;kidney or renal cancer; larynx cancer; leukemia; liver cancer; lungcancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue,mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as wellas B-cell lymphoma (including low grade/follicular non-Hodgkin'slymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade/follicular NHL; intermediate grade diffuse NHL; high gradeimmunoblastic NHL; high grade lymphoblastic NHL; high grade smallnon-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (e.g. that associated with brain tumors), andMeigs' syndrome.

In some embodiments, the present invention relates to the treatment of,or a patient having a microbial infection and/or chronic infection.Illustrative infections include, but are not limited to, HIV/AIDS,tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barrvirus or parvovirus, T cell leukemia virus, bacterial overgrowthsyndrome, fungal or parasitic infections.

In various embodiments, the present compositions are used to treat orprevent one or more inflammatory diseases or conditions, such asinflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopicdermatitis, septic shock, rheumatoid arthritis, inflammatory boweldisease, inflammatory pelvic disease, pain, ocular inflammatory disease,celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familialeosinophilia (FE), autosomal recessive spastic ataxia, laryngealinflammatory disease; Tuberculosis, Chronic cholecystitis,Bronchiectasis, Silicosis and other pneumoconioses.

In various embodiments, the present compositions are used to treat orprevent one or more autoimmune diseases or conditions, such as multiplesclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease,ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture'ssyndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen'sencephalitis, Primary biliary sclerosis, Sclerosing cholangitis,Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis,Fibromyalgia, Menier's syndrome; transplantation rejection (e.g.,prevention of allograft rejection) pernicious anemia, rheumatoidarthritis, systemic lupus erythematosus, dermatomyositis, Sjogren'ssyndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis,Reiter's syndrome, Grave's disease, and other autoimmune diseases.

In various embodiments, the present compositions are used to treat,control or prevent cardiovascular disease, such as a disease orcondition affecting the heart and vasculature, including but not limitedto, coronary heart disease (CHD), cerebrovascular disease (CVD), aorticstenosis, peripheral vascular disease, atherosclerosis,arteriosclerosis, myocardial infarction (heart attack), cerebrovasculardiseases (stroke), transient ischaemic attacks (TIA), angina (stable andunstable), atrial fibrillation, arrhythmia, vavular disease, and/orcongestive heart failure.

In various embodiments, the present compositions are used to treat orprevent one or more metabolic-related disorders. In various embodiments,the present invention is useful for the treatment, controlling orprevention of diabetes, including Type 1 and Type 2 diabetes anddiabetes associated with obesity. The compositions and methods of thepresent invention are useful for the treatment or prevention ofdiabetes-related disorders, including without limitation diabeticnephropathy, hyperglycemia, impaired glucose tolerance, insulinresistance, obesity, lipid disorders, dyslipidemia, hyperlipidemia,hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDLlevels, atherosclerosis and its sequelae, vascular restenosis, irritablebowel syndrome, inflamatory bowel disease, including Crohn's disease andulcerative colitis, other inflammatory conditions, pancreatitis,abdominal obesity, neurodegenerative disease, retinopathy, neoplasticconditions, adipose cell tumors, adipose cell carcinomas, such asliposarcoma, prostate cancer and other cancers, including gastric,breast, bladder and colon cancers, angiogenesis, Alzheimer's disease,psoriasis, high blood pressure, Metabolic Syndrome (e.g. a person hasthree or more of the following disorders: abdominal obesity,hypertriglyceridemia, low HDL cholesterol, high blood pressure, and highfasting plasma glucose), ovarian hyperandrogenism (polycystic ovarysyndrome), and other disorders where insulin resistance is a component,such as sleep apnea. The compositions and methods of the presentinvention are useful for the treatment, control, or prevention ofobesity, including genetic or environmental, and obesity-relateddisorders. The obesity-related disorders herein are associated with,caused by, or result from obesity. Examples of obesity-related disordersinclude obesity, diabetes, overeating, binge eating, and bulimia,hypertension, elevated plasma insulin concentrations and insulinresistance, dyslipidemia, hyperlipidemia, endometrial, breast, prostate,kidney and colon cancer, osteoarthritis, obstructive sleep apnea,gallstones, heart disease, abnormal heart rhythms and arrythmias,myocardial infarction, congestive heart failure, coronary heart disease,sudden death, stroke, polycystic ovary disease, craniopharyngioma,Prader-Willi Syndrome, Frohlich's syndrome, GH-deficient subjects,normal variant short stature, Turner's syndrome, and other pathologicalconditions showing reduced metabolic activity or a decrease in restingenergy expenditure as a percentage of total fat-free mass, e.g, childrenwith acute lymphoblastic leukemia. Further examples of obesity-relateddisorders are Metabolic Syndrome, insulin resistance syndrome,reproductive hormone abnormalities, sexual and reproductive dysfunction,such as impaired fertility, infertility, hypogonadism in males andhirsutism in females, fetal defects associated with maternal obesity,gastrointestinal motility disorders, such as obesity-relatedgastro-esophageal reflux, respiratory disorders, such asobesity-hypoventilation syndrome (Pickwickian syndrome), breathlessness,cardiovascular disorders, inflammation, such as systemic inflammation ofthe vasculature, arteriosclerosis, hypercholesterolemia, lower backpain, gallbladder disease, hyperuricemia, gout, and kidney cancer, andincreased anesthetic risk. The compositions and methods of the presentinvention are also useful to treat Alzheimer's disease.

In various embodiments, the present compositions are used to treat orprevent one or more respiratory diseases, such as asthma, chronicobstructive pulmonary disease (COPD), bronchiectasis, allergic rhinitis,sinusitis, pulmonary vasoconstriction, inflammation, allergies, impededrespiration, respiratory distress syndrome, cystic fibrosis, pulmonaryhypertension, pulmonary vasoconstriction, emphysema, Hantaviruspulmonary syndrome (HPS), Loeffler's syndrome, Goodpasture's syndrome,Pleurisy, pneumonitis, pulmonary edema, pulmonary fibrosis, Sarcoidosis,complications associated with respiratory syncitial virus infection, andother respiratory diseases.

In some embodiments, the present invention is used to treat or preventone or more neurodegenerative disease. Illustrative neurodegenerativedisease include, but are not limited to, multiple sclerosis (includingwithout limitation, benign multiple sclerosis; relapsing-remittingmultiple sclerosis (RRMS); secondary progressive multiple sclerosis(SPMS); progressive relapsing multiple sclerosis (PRMS); and primaryprogressive multiple sclerosis (PPMS)), Alzheimer's. disease (including,without limitation, Early-onset Alzheimer's, Late-onset Alzheimer's, andFamilial Alzheimer's disease (FAD), Parkinson's disease and parkinsonism(including, without limitation, Idiopathic Parkinson's disease, Vascularparkinsonism, Drug-induced parkinsonism, Dementia with Lewy bodies,Inherited Parkinson's, Juvenile Parkinson's), Huntington's disease,Amyotrophic lateral sclerosis (ALS, including, without limitation,Sporadic ALS, Familial ALS, Western Pacific ALS, Juvenile ALS, HiramayaDisease).

In various embodiments, the present chimeric proteins find use intreating wounds, e.g., a non-healing wound, an ulcer, a burn, orfrostbite, a chronic or acute wound, open or closed wound, internal orexternal wound (illustrative external wounds are penetrating andnon-penetrating wound. In various embodiments, the present chimericproteins find use in treating ischemia, by way of non-limiting example,ischemia associated with acute coronary syndrome, acute lung injury(ALI), acute myocardial infarction (AMI), acute respiratory distresssyndrome (ARDS), arterial occlusive disease, arteriosclerosis, articularcartilage defect, aseptic systemic inflammation, atheroscleroticcardiovascular disease, autoimmune disease, bone fracture, bonefracture, brain edema, brain hypoperfusion, Buerger's disease, burns,cancer, cardiovascular disease, cartilage damage, cerebral infarct,cerebral ischemia, cerebral stroke, cerebrovascular disease,chemotherapy-induced neuropathy, chronic infection, chronic mesentericischemia, claudication, congestive heart failure, connective tissuedamage, contusion, coronary artery disease (CAD), critical limb ischemia(CLI), Crohn's disease, deep vein thrombosis, deep wound, delayed ulcerhealing, delayed wound-healing, diabetes (type I and type II), diabeticneuropathy, diabetes induced ischemia, disseminated intravascularcoagulation (DIC), embolic brain ischemia, frostbite, graft-versus-hostdisease, hereditary hemorrhagic telengiectasiaischemic vascular disease,hyperoxic injury, hypoxia, inflammation, inflammatory bowel disease,inflammatory disease, injured tendons, intermittent claudication,intestinal ischemia, ischemia, ischemic brain disease, ischemic heartdisease, ischemic peripheral vascular disease, ischemic placenta,ischemic renal disease, ischemic vascular disease, ischemic-reperfusioninjury, laceration, left main coronary artery disease, limb ischemia,lower extremity ischemia, myocardial infarction, myocardial ischemia,organ ischemia, osteoarthritis, osteoporosis, osteosarcoma, Parkinson'sdisease, peripheral arterial disease (PAD), peripheral artery disease,peripheral ischemia, peripheral neuropathy, peripheral vascular disease,pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorder,renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers,solid organ transplantation, spinal cord injury, stroke,subchondral-bone cyst, thrombosis, thrombotic brain ischemia, tissueischemia, transient ischemic attack (TIA), traumatic brain injury,ulcerative colitis, vascular disease of the kidney, vascularinflammatory conditions, von Hippel-Lindau syndrome, or wounds totissues or organs

In various embodiments, the present invention relates to the treatmentof one or more of anemia, including anemia resulting from chronic kidneydisease (e.g. from dialysis) and/or an anti-cancer agent (e.g.chemotherapy and/or HIV treatment (e.g. Zidovudine (INN) orazidothymidine (AZT)), inflammatory bowel disease (e.g. Crohn's diseaseand ulcer colitis), anemia linked to inflammatory conditions (e.g.arthritis, lupus, IBD), anemia linked to diabetes, schizophrenia,cerebral malaria, as aplastic anemia, and myelodysplasia from thetreatment of cancer (e.g. chemotherapy and/or radiation), and variousmyelodysplastic syndrome diseases (e.g. sickle cell anemia, hemoglobinSC disease, hemoglobin C disease, alpha- and beta-thalassemias, neonatalanemia after premature birth, and comparable conditions).

In some embodiments, the present invention relates to the treatment of,or a patient having anemia, i.e. a condition in which the number of redblood cells and/or the amount of hemoglobin found in the red blood cellsis below normal. In various embodiments, the anemia may be acute orchronic. For example, the present anemias include but are not limited toiron deficiency anemia, renal anemia, anemia of chronicdiseases/inflammation, pernicious anemia such as macrocytic achylicanemia, juvenile pernicious anemia and congenital pernicious anemia,cancer-related anemia, anti-cancer-related anemia (e.g.chemotherapy-related anemia, radiotherapy-related anemia), pure red cellaplasia, refractory anemia with excess of blasts, aplastic anemia,X-lined siderobalstic anemia, hemolytic anemia, sickle cell anemia,anemia caused by impaired production of ESA, myelodysplasia syndromes,hypochromic anemia, microcytic anemia, sideroblastic anemia, autoimmunehemolytic anemia, Cooley's anemia, Mediterranean anemia, DiamondBlackfan anemia, Fanconi's anemia and drug-induced immune hemolyticanemia. Anemia may cause serious symptoms, including hypoxia, chronicfatigue, lack of concentration, pale skin, low blood pressure, dizzinessand heart failure.

In some embodiments, the present invention relates to the treatment ofanemia resulting from chronic renal failure. In some embodiments, thepresent invention relates to the treatment of anemia resulting from theuse of one or more renal replacement therapies, inclusive of dialysis,hemodialysis, peritoneal dialysis, hemofiltration, hemodiafiltration,and renal transplantation.

In some embodiments, the present invention relates to the treatment ofanemia in patients with chronic kidney disease who are not on dialysis.For instance, the present invention relates to patients in stage 1 CKD,or stage 2 CKD, or stage 3 CKD, or stage 4 CKD, or stage 5 CKD. In someembodiments, the present patient is stage 4 CKD or stage 5 CKD. In someembodiments, the present patient has undergone a kidney transplant. Insome embodiments, the present invention relates to the treatment ofanemia is a patient having an acute kidney injury (AKI).

In some embodiments, the anemia is induced by chemotherapy. Forinstance, the chemotherapy may be any myelosuppressive chemotherapy. Insome embodiment, the chemotherapy is one or more of Revlimid, Thalomid,dexamethasone, Adriamycin and Doxil. In some embodiments, thechemotherapy is one or more platinum-based drugs including cisplatin(e.g. PLATINOL) and carboplatin (e.g. PARAPLATIN). In some embodiments,the chemotherapy is any one of the chemotherapeutic agents describedherein. In some embodiments, the chemotherapy is any agent described inGroopman et al. J Natl Cancer Inst (1999) 91 (19): 1616-1634, thecontents of which are hereby incorporated by reference in theirentireties. In some embodiments, the present compositions and methodsare used in the treatment of chemotherapy-related anemia in later stagecancer patients (e.g. a stage IV, or stage III, or stage II cancer). Insome embodiments, the present compositions and methods are used in thetreatment of chemotherapy-related anemia in cancer patients receivingdose-dense chemotherapy or other aggressive chemotherapy regimens.

In some embodiments, the present invention relates to the treatment ofanemia in a patient having one or more blood-based cancers, such asleukemia, lymphoma, and multiple myeloma. Such cancers may affect thebone marrow directly. Further, the present invention relates tometastatic cancer that has spread to the bone or bone marrow. In someembodiments, the present invention relates to the treatment of anemia ina patient undergoing radiation therapy. Such radiation therapy maydamage the bone marrow, lowering its ability to make red blood cells. Infurther embodiments, the present invention relates to the treatment ofanemia in a patient having a reduction or deficiency of one or more ofiron, vitamin B12, and folic acid. In further embodiments, the presentinvention relates to the treatment of anemia in a patient havingexcessive bleeding including without limitation, after surgery or from atumor that is causing internal bleeding. In further embodiments, thepresent invention relates to the treatment of anemia in a patient havinganemia of chronic disease.

In some embodiments, the present methods and compositions stimulate redblood cell production. In some embodiments, the present methods andcompositions stimulate division and differentiation of committederythroid progenitors in the bone marrow.

Certain embodiments of the present invention are particularly useful fortreating chemotherapy-induced anemia in cancer patients. In someembodiments, the present methods and compositions allows for continuedadministration of the chimeric protein after a cancer patient'schemotherapy is finished. In some embodiments, the present methods andcompositions allows for treatment of a cancer patient without dosereduction relative to a non-cancer patient. In some embodiments, thepresent methods and compositions allows for treatment of a cancerpatient receiving chemotherapy and considered curable. In variousembodiments, the cancer patient has one or more of a history of bloodclots, recent surgery, prolonged periods of bed rest or limitedactivity, and treatment with a chemotherapeutic agent.

Kits

The invention also provides kits for the administration of any agentdescribed herein (e.g. the chimeric protein with or without variousadditional therapeutic agents). The kit is an assemblage of materials orcomponents, including at least one of the inventive pharmaceuticalcompositions described herein. Thus, in some embodiments, the kitcontains at least one of the pharmaceutical compositions describedherein.

The exact nature of the components configured in the kit depends on itsintended purpose. In one embodiment, the kit is configured for thepurpose of treating human subjects.

Instructions for use may be included in the kit. Instructions for usetypically include a tangible expression describing the technique to beemployed in using the components of the kit to effect a desired outcome,such as to treat anemia. Optionally, the kit also contains other usefulcomponents, such as, diluents, buffers, pharmaceutically acceptablecarriers, syringes, catheters, applicators, pipetting or measuringtools, bandaging materials or other useful paraphernalia as will bereadily recognized by those of skill in the art.

The materials and components assembled in the kit can be provided to thepractitioner stored in any convenience and suitable ways that preservetheir operability and utility. For example, the components can beprovided at room, refrigerated or frozen temperatures. The componentsare typically contained in suitable packaging materials. In variousembodiments, the packaging material is constructed by well-knownmethods, preferably to provide a sterile, contaminant-free environment.The packaging material may have an external label which indicates thecontents and/or purpose of the kit and/or its components.

Definitions

As used herein, “a,” “an,” or “the” can mean one or more than one.

Further, the term “about” when used in connection with a referencednumeric indication means the referenced numeric indication plus or minusup to 10% of that referenced numeric indication. For example, thelanguage “about 50” covers the range of 45 to 55.

An “effective amount,” when used in connection with medical uses is anamount that is effective for providing a measurable treatment,prevention, or reduction in the rate of pathogenesis of a disease ofinterest.

As used herein, something is “decreased” if a read-out of activityand/or effect is reduced by a significant amount, such as by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100%, inthe presence of an agent or stimulus relative to the absence of suchmodulation. As will be understood by one of ordinary skill in the art,in some embodiments, activity is decreased and some downstream read-outswill decrease but others can increase.

Conversely, activity is “increased” if a read-out of activity and/oreffect is increased by a significant amount, for example by at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, or more, up to and including at least about 100% ormore, at least about 2-fold, at least about 3-fold, at least about4-fold, at least about 5-fold, at least about 6-fold, at least about7-fold, at least about 8-fold, at least about 9-fold, at least about10-fold, at least about 50-fold, at least about 100-fold, in thepresence of an agent or stimulus, relative to the absence of such agentor stimulus.

As referred to herein, all compositional percentages are by weight ofthe total composition, unless otherwise specified. As used herein, theword “include,” and its variants, is intended to be non-limiting, suchthat recitation of items in a list is not to the exclusion of other likeitems that may also be useful in the compositions and methods of thistechnology. Similarly, the terms “can” and “may” and their variants areintended to be non-limiting, such that recitation that an embodiment canor may comprise certain elements or features does not exclude otherembodiments of the present technology that do not contain those elementsor features.

Although the open-ended term “comprising,” as a synonym of terms such asincluding, containing, or having, is used herein to describe and claimthe invention, the present invention, or embodiments thereof, mayalternatively be described using alternative terms such as “consistingof” or “consisting essentially of.”

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology.

The amount of compositions described herein needed for achieving atherapeutic effect may be determined empirically in accordance withconventional procedures for the particular purpose. Generally, foradministering therapeutic agents for therapeutic purposes, thetherapeutic agents are given at a pharmacologically effective dose. A“pharmacologically effective amount,” “pharmacologically effectivedose,” “therapeutically effective amount,” or “effective amount” refersto an amount sufficient to produce the desired physiological effect oramount capable of achieving the desired result, particularly fortreating the disorder or disease. An effective amount as used hereinwould include an amount sufficient to, for example, delay thedevelopment of a symptom of the disorder or disease, alter the course ofa symptom of the disorder or disease (e.g., slow the progression of asymptom of the disease), reduce or eliminate one or more symptoms ormanifestations of the disorder or disease, and reverse a symptom of adisorder or disease. Therapeutic benefit also includes halting orslowing the progression of the underlying disease or disorder,regardless of whether improvement is realized.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to about 50% ofthe population) and the ED50 (the dose therapeutically effective inabout 50% of the population). The dosage can vary depending upon thedosage form employed and the route of administration utilized. The doseratio between toxic and therapeutic effects is the therapeutic index andcan be expressed as the ratio LD50/ED50. In some embodiments,compositions and methods that exhibit large therapeutic indices arepreferred. A therapeutically effective dose can be estimated initiallyfrom in vitro assays, including, for example, cell culture assays. Also,a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the 1050 as determined in cellculture, or in an appropriate animal model. Levels of the describedcompositions in plasma can be measured, for example, by high performanceliquid chromatography. The effects of any particular dosage can bemonitored by a suitable bioassay. The dosage can be determined by aphysician and adjusted, as necessary, to suit observed effects of thetreatment.

In certain embodiments, the effect will result in a quantifiable changeof at least about 10%, at least about 20%, at least about 30%, at leastabout 50%, at least about 70%, or at least about 90%. In someembodiments, the effect will result in a quantifiable change of about10%, about 20%, about 30%, about 50%, about 70%, or even about 90% ormore. Therapeutic benefit also includes halting or slowing theprogression of the underlying disease or disorder, regardless of whetherimprovement is realized.

As used herein, “methods of treatment” are equally applicable to use ofa composition for treating the diseases or disorders described hereinand/or compositions for use and/or uses in the manufacture of amedicaments for treating the diseases or disorders described herein.This invention is further illustrated by the following non-limitingexamples.

EXAMPLES

The term “AcTaferon” is occasionally used herein to reference aninterferon-based chimera.

In the following examples, unless noted, mutations to IFN are relativeto human IFN-α2—SEQ ID NO: 336.

The Q124R mutant is representative of an attenuated human IFN alpha 2mutant that can be assayed in vivo in a murine model. Specifically,Q124R is a human IFN mutation that is suitable for use in the mouse(i.e. it is a human mutant IFN that functions in mouse). See Nat. Comm.2014; 5:3016. doi: 10.1038/ncomms4016, the entire contents of which arehereby incorporated by reference.

The R33A/E120R mutant is representative of human IFN alpha 2 mutant thatis non-functional (and is used as a control)

Anti-human PD-1 VHH used in these Examples is SEQ ID NO: 132.

Anti-human PD-L1 VHH used in these Examples is SEQ ID NO: 267.

Anti-human CLEC9A VHH used in these Examples is R2CHCL24 (SEQ ID NO: 87)Anti-human CD3 scFV used in these Examples is SEQ ID NO: 365.

Anti-human CD20 scFV used in these Examples is SEQ ID NO: 366.

Anti-human CD8 VHH used in these Examples is R2HCD26 (SEQ ID NO:21).

Anti-Bcll10 VHH is used in these Examples as a control (targeting anirrelevant antigen, i.e. “untargeted”).

Example 1: Characterization of Bispecific Chimeras Using an In Vivo B16Melanoma Model

Various bispecific chimeric constructs were engineered with anti-mousePD-L1, anti-mouse CD20, anti-mouse Clec9A, and/or anti-mouse CD8targeting moieties and a modified interferon signaling domain (i.e., themutant human interferon (IFN) α2-Q124R or R149A). A list of the chimericconstructs is provided below. The in vivo activities of the chimera werecharacterized using an in vivo B16 melanoma model.

PBS BCII.10-Q124R (10120) PD-L1-Q124R (12246) CLEC9A-Q124R (10666)CLEC9A-R149A (10685) CLEC9A-Q124R-PD-L1 (11645) CLEC9A-R149A-PD-L1(13305) PD-L1-Q124R-CLEC9A (12938) PD-L1-R149A-CLEC9A CLEC9A-PD-L1-Q124R(13271) CLEC9A-PD-L1-R149A (13331) PD-L1-CLEC9A-Q124R (13272)PD-L1-CLEC9A-R149A (13332) CD8-Q124R (11487) CD8-R149A (13339)CD8-Q124R-PD-L1 (12277) CD8-R149A-PD-L1 (13306) PD-L1-Q124R-CD8 (12939)PD-L1-R149A-CD8 CD8-PD-L1-Q124R (13309) CD8-PD-L1-R149A (13337)PD-L1-CD8-Q124R (13310) PD-L1-CD8-R149A (13338) CLEC9A-Q124R-CD8 (12942)CLEC9A-R149A-CD8 CD8-Q124R-CLEC9A (12941) CD8-R149A-CLEC9ACLEC9A-CD8-Q124R (13276) CLEC9A-CD8-R149A CD8-CLEC9A-Q124R (13308)CD8-CLEC9A-R149A

FIG. 1 shows the anti-tumor activities of an anti-CD20 VHH-humanIFNalpha (Q124R) mutant-anti-PD-L1 VHH bispecific construct using a B16tumor model. In the experiment, C57BL/6 mice were inoculatedsubcutaneously (50 μl) with 6×10⁵ B16mCD20cl1 melanoma tumor cells (amouse melanoma cell line that stably expresses mouse CD20). Perilesionaltreatment with 30 μg of various constructs (100 μl) was started whentumors reached a size of ±10 mm² as measured by caliper. The constructstested included a monospecific anti-PD-L1 VHH, a monospecific anti-PD-L1VHH-human IFNalpha, Q124R mutant construct, a monospecific anti-CD20VHH-human IFNalpha, Q124R mutant construct, and the bispecific anti-CD20VHH-human IFNalpha, Q124R mutant-anti-PD-L1 VHH construct. FIG. 1, panelA, shows that anti-PD-L1 had no effect in the B16 tumor model while themonospecific PD-L1-Q124R construct showed some anti-tumor activity andreduced tumor size. Panel B shows that a bispecific chimera that targetsboth CD20 and PD-L1 (“mCD20-Q124R-PD-L1”) was superior to a monospecificchimera that only targets CD20 (“mCD20-Q124R”) with respect toanti-tumor activity (panel B). In both the bispecific and monospecificconstructs, a modified interferon signaling domain was present (Q124R).

FIG. 2 shows the anti-tumor activities of a bispecific chimera(“Clec9A-Q124R-PD-L1”) using the B16 tumor model described above. Asshown in FIG. 2, the bispecific chimera Clec9A-Q124R-PD-L1 was superiorto a monospecific anti-Clec9a chimera (“Clec9A-Q124R”) and PD-L1(“PD-L1-Q124R”) chimera. All of the chimeras were fused to modifiedinterferon: Q124R.

Both mCD20-Q124R-PD-L1 and Clec9A-Q124R-PD-L1 bi-specific constructswere shown to be safe (FIG. 3 and FIG. 4). In each figure, panels A-Gshow: change in body weight of the mice in the tumor studies describedabove (panel A), white blood cell counts (“wbc”) and lymphocytes count(“ly”) (panel B), neutrophil count (“ne”) and monocyte count (“mo”)(panel C); red blood cell count (“rbc”) and, for FIG. 4, hemoglobin(“hb”) (panel D); hemocrit (“hct”), mean corpuscular volume (“mcv”),mean corpuscular hemoglobin (“mch”), mean corpuscular hemoglobinconcentration (“mchc”) (panel E); pitted red blood cells (“pit”) (panelF); and mean platelet volume (“mpv”) (panel G).

FIG. 5 shows a B16 tumor model study in which a bi-specific (anti-Clec9Aand anti-PD-L1) fusion to modified human IFN alpha (Q124R) providedbetter anti-tumor activity as compared to the co-administration of afusion of anti-Clec9A to modified human IFN alpha (Q124R) and a fusionof anti-PD-L1 to modified human IFN alpha (Q124R) or theco-administration of a fusion of anti-Clec9A to modified human IFN alpha(Q124R) and an anti-PD-L1 VHH.

The anti-tumor activities of a bispecific chimeric (“CD8-Q124R-PD-L1”)was also analyzed using the B16 melanoma model. As shown in FIG. 6, thebi-specific (anti-CD8 and anti-PD-L1) fusion to modified human IFN alpha(Q124R) provided better anti-tumor activity as compared to a fusion ofanti-CD8 to modified human IFN alpha (Q124R).

A Batf3 knockout (Batf3−/−) mouse model was utilized to study the roleof cross-presenting dendritic cells in tumorigenesis and the anti-tumoractivities of the bispecific constructs. The Batf3 −/− C57BL6 mice lackthe Batf3 gene and thus do not have CD8a+ cross-presenting dendriticcells. In the experiment, Batf3 −/− mice were inoculated subcutaneouslywith B16mCD20cl1 melanoma tumor cells. When tumors reached a certainsize, the mice were then treated with a bi-specific (anti-Clec9A andanti-PD-L1) fusion to modified human IFN alpha (Q124R). As shown in FIG.7, the Batf3 −/− mice were more susceptible to tumor formation andformed larger tumors than wild type C57BL6 mice. Administration of thebispecific (anti-Clec9A and anti-PD-L1) fusion to modified human IFNalpha (Q124R) reduced tumor size in both Batf3 −/− and wild type C57BL6mice. Further, without wishing to be bound by theory, comparing theeffectiveness of the bispecific (anti-Clec9A and anti-PD-L1) fusion tomodified human IFN alpha (Q124R) in the two genetic backgroundsunderscores the importance of dendritic cells for Clec9A targeting.

Example 2: Characterization of Bispecific Chimeras Using an In Vivo 4T1Mammary Tumor Model

The antitumor activities of the various mono-specific and bi-specificchimeric constructs were also tested using a 4T1 mammary tumor model. Inthe experiment, mice were inoculated with 4T1 mammary tumor cells. Oncetumors reached a certain size, the mice were treated with a monospecificanti-PD-L1 VHH or a monospecific anti-PD-L1 VHH-human IFNalpha, Q124Rmutant construct. As shown in FIG. 8, panel A, anti-PD-L1 has no effectin the 4T1 mammary tumor model while the monospecific PD-L1-Q124Rconstruct had anti-tumor activity.

FIG. 8, panel B shows a 4T1 mammary tumor model study in which abi-specific (anti-Clec9A and anti-PD-L1) fusion to modified human IFNalpha (Q124R) provided better anti-tumor activity as compared to theco-administration of a fusion of anti-Clec9A to modified human IFN alpha(Q124R) and a fusion of anti-PD-L1 to modified human IFN alpha (Q124R)or the co-administration of a fusion of anti-Clec9A to modified humanIFN alpha (Q124R) and an anti-PD-L1 VHH.

FIG. 8, panel C shows a 4T1 mammary tumor model study in which abi-specific (anti-CD8 and anti-PD-L1) fusion to modified human IFN alpha(Q124R) provided better anti-tumor activity as compared to theco-administration of a fusion of anti-CD8 to modified human IFN alpha(Q124R) and a fusion of anti-PD-L1 to modified human IFN alpha (Q124R)or the co-administration of a fusion of anti-CD8 to modified human IFNalpha (Q124R) and an anti-PD-L1 VHH.

Example 3: Combination Therapy Using Bispecific Chimera and Doxorubicinin the B16 Melanoma and 4T1 Mammary Tumor Models

The anti-tumor effects of a combination therapy using doxorubicin andbispecific constructs were tested. In one set of experiments, mice weretransplanted with B16 cells (a mouse melanoma cell line that stablyexpresses mouse CD20) to induce tumors. In a second set of experiments,the mice were transplanted with 4T1 mammary tumor cells. The mice weresubsequently treated with a bi-specific (anti-mouse Clec9A andanti-mouse PD-L1) fusion to modified human IFN alpha (Q124R) with orwithout doxorubicin. In a third set of experiment, the mice weretransplanted with 4T1 mammary tumor cells and subsequently treated witha bi-specific (anti-CD8 and anti-PD-L1) fusion to modified human IFNalpha (Q124R) with or without doxorubicin. As shown in FIG. 9, panelsA-C, in all three tumor models, combining the bispecific chimera withdoxorubicin significantly reduced tumor size compared with treatmentusing the bispecific chimera alone or doxorubicin alone. Notably asshown in FIG. 9, panel B, in the 4T1 mammary tumor model, the use of thebi-specific (anti-Clec9A and anti-PD-L1) fusion to modified human IFNalpha (Q124R) with doxorubicin resulted in a curative effect in 5 out of6 mice (i.e., the mice were completely tumor free). Similarly, as shownin FIG. 9, panel C, in the 4T1 mammary tumor model, the use of thebi-specific (anti-CD8 and anti-PD-L1) fusion to modified human IFN alpha(Q124R) with doxorubicin also resulted in a curative effect in 3 out of6 mice (i.e., the mice were completely tumor free). Without wishing tobe bound by theory, it is believed that the combination of thebispecific agent and doxorubicin resulted in a synergistic effect (i.e.,complete eradiation of tumors) compared to use of the agentsindividually.

The 4T1 tumor model was utilized conduct a tumor re-challengeexperiment, in which mice that remained tumor free (i.e., the 5 micethat were tumor free as demonstrated in FIG. 9, panel B) werere-challenged with additional 4T1 tumor cells. Specifically, in theexperiment, mice were initially inoculated with 4T1 mammary tumor cellsand treated with a combination of doxorubicin and a fusion ofanti-Clec9A to modified human IFN alpha (Q124R) or a bi-specific(anti-Clec9A and anti-PD-L1) fusion to modified human IFN alpha (Q124R)as described previously (for example, in FIG. 9). Mice that remainedtumor free were then re-challenged with 10⁵ 4 T1 tumor cells andmonitored for tumor growth without any additional treatment.

Without wishing to be bound by theory, FIG. 10 shows that the presentbi-specific (anti-Clec9A and anti-PD-L1) fusion to modified human IFNalpha (Q124R) provided an anti-tumor memory effect.

Example 4: Efficacy of Bispecific Chimera in a Mouse Model of MultipleSclerosis

The efficacy of the bispecific constructs in treating multiple sclerosiswas tested using an established mouse experimental autoimmuneencephalomyelitis (EAE) model. Specifically, the mice weresubcutaneously administered peptides corresponding to the immunodominantepitopes of MOG (MOG₃₅₋₅₅) suspended in complete Freund's adjuvant (CFA)derived from killed Mycobacterium tuberculosis bacilli (Mtb). Pertussistoxin was also administered to the mice by intraperitoneal injections onthe day of and two days later. The mice were then treated from days 7-25with either 100 IU or 1000 IU of a monospecific fusion of anti-mouseClec9A to modified human IFN alpha Q124R (“Clec9A-Q124R”) or amonospecific fusion of anti-mouse mCD20 to modified human IFN alphaQ124R (“mCD20-Q124R”) chimera or a combination of both mono-specificchimeras. Alternatively, the mice were treated with either 100 IU or1000 IU of a Clec9A-Q124R-mCD20 (i.e. bi1) or Clec-9A-mCD20-Q124R (i.e.bi2) bispecific chimera, which differ in the configuration of thetargeting moieties (i.e., the anti-Clec9A and anti-mCD20 VHHs) and thesignaling agent (i.e., IFNalpha, Q124R mutant). The mice werephenotypically scored on a daily basis as depicted in FIG. 11, panel A.

As shown in FIG. 11, panel B, at the 100 IU dosage, theClec-9A-mCD20-Q124R bispecific chimera significantly improved clinicalscores compared to treatment with either of the monospecific chimeras ora combination of the monospecific chimeras. The Clec-9A-mCD20-Q124Rbispecific chimera also exhibited superior activities compared to theClec9A-Q124R-mCD20 bispecific chimera. Similarly, at the 1000 IU dosagelevel, the Clec-9A-mCD20-Q124R bispecific chimera significantly improvedclinical scores compared to treatment with either of the monospecificchimeras or a combination of the monospecific chimeras (FIG. 11, panelC). At this dosage level, the Clec9A-Q124R-mCD20 bispecific chimera alsoexhibited superior activities compared to treatment with either of themonospecific chimeras or a combination of the monospecific chimeras(FIG. 11, panel C). Additionally, it was shown that theClec-9A-mCD20-Q124R bispecific chimera was safe to use at the 1000 IUdosage level. As shown in FIG. 11, panel D, at this dosage, thebispecific chimera did not induce lymphopenia in treated mice.

In summary, as further shown in FIG. 11, panel E, the Clec9A-Q124R-mCD20bispecific chimera provided superior protection against multiplesclerosis in the EAE model at a dosage of 1000 IU. In comparison, theClec-9A-mCD20-Q124R bispecific chimera provided superior protectionagainst multiple sclerosis in the EAE model at both the 100 IU and 1000IU dosage levels and induced no lymphopenia in treated mice (FIG. 11,panel F). Notably, in mice treated with 100 IU of theClec-9A-mCD20-Q124R bispecific chimera (i.e., bi2), 1 out 5 mice did notshow any disease symptoms.

Experiments were also carried out to assess the efficacy of theClec-9A-mCD20-Q124R bispecific chimera at the 5000 IU dosage level. Inthese experiments, a control Clec-9A-mCD20-R149A bispecific chimera wasalso used. In these experiments, the bispecific chimeras wereadministered starting at day 7 or day 12 and continued until day 25.

As shown in FIG. 12, panel A, the Clec-9A-mCD20-Q124R bispecific chimerasignificantly improved clinical scores compared to controls whenadministered at either day 7 or day 12 at a dosage level of 5000 IU.More specifically, when the bispecific chimera was administered beforedisease onset, i.e., at day 7, disease onset was delayed or stoppedaltogether. Importantly, when the bispecific chimera was administeredafter disease onset, i.e. at day 12, disease progression was slowed.Without wishing to be bound by theory, administration of the bispecificchimera can be utilized for both prevention and treatment (e.g., slowingprogression) of multiple sclerosis. The bispecific chimera also appearedto be safe to use at this dosage level as the treated mice did not showsignificant loss in body weight up to day 20 of treatment (FIG. 12,panel B). As shown in FIG. 12, panel C, the Clec-9A-mCD20-Q124Rbispecific chimera also significantly delayed disease onset andincidence compared to controls.

Example 5. Tumor-Antigen Specific CD8⁺ T Cell Proliferation andActivation with Bi-Specific Chimeras

FIG. 13 shows an evaluation of tumor-antigen specific CD8⁺ T cellproliferation and activation both in tumor-draining lymph nodes as wellas in spleen, two prime organs in the induction of anti-tumor immunity.

Proliferation of Tumor-Antigen Specific T Cells

To evaluate proliferation of tumor-antigen specific T cells, T cellreceptor transgenic CD8⁺ T cells (OT-I cells) were utilized whichspecifically recognized the model antigen ovalbumin (OVA) present onB16-OVA tumor cells. CD8⁺ T cells were isolated from the spleens ofC57BL/6 OT-I mice using the CD8a T cell isolation kit (Miltenyi Biotec)and labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE,Thermo Fisher). One million of CFSE-labeled CD8 purified OT-I cells wereadoptively transferred to C57BL/6 mice inoculated 9 days earlier with6×10⁵ B16-OVA melanoma cells expressing ovalbumin. One day after OT-Itransfer, mice were treated perilesional with PBS or with 30 μgbispecific chimera of anti-Clec9A VHH/anti-PD-L1 VHH/human IFN Q124R.Some mice were additionally injected perilesionaly with doxorubicin (3mg/kg, Sigma). Five days post adoptive OT-I transfer, tumor-draininglymph nodes and spleen were isolated and OT-I CD8 T cell proliferationwas assessed by flow cytometry (FIG. 13, panels A and C). Samples wereacquired on an Attune Nxt Acoustic Focusing Cytometer (LifeTechnologies) and analyzed using FlowJo software.

Activation of Tumor-Antigen Specific T Cells

To analyze the phenotype of the proliferated OT-I CD8 T cells, singlecell suspension of tumor-draining lymph nodes and spleens were stainedfor different T cell activation markers. Therefore, Fc receptors wereblocked using CD16/CD32 antibody (eBioscience) prior to staining withCD8-APC (clone 53-6.7, BD Pharmingen), CD44-PercP-Cy5.5 (clone IMF7,Biolegend) and CD62L-APC-Cy7 (clone MEL-14, Biolegend). Gating strategywas first performed on CD8⁺ cells of which the CFSE diluting(proliferating) T cells were selected. Naive T cells were based onCD44^(low) CD62L^(high) phenotype, effector T cells on CD44^(high)CD62L^(low) phenotype and memory T cells based on CD44^(high)CD62L^(high) phenotype (FIG. 13, panels B and D). Samples were acquiredon an Attune Nxt Acoustic Focusing Cytometer (Life Technologies) andanalyzed using FlowJo software.

It is well established that CD8⁺ cytotoxic T lymphocytes play a crucialrole in eradicating tumor cells. The data of this Example shows, interalia, that the bispecific chimera of anti-Clec9A VHH/anti-PD-L1VHH/human IFN Q124R induced tumor-antigen specific CD8⁺ T cellproliferation and activation both in tumor-draining lymph nodes (FIG.13, panels A and B) as well as in spleen (FIG. 13, panels C and D).

Example 6. Sirp1α and DNAM Bi-Specific Chimeras

The efficiency of mouse PD-L1 targeting by various bi-specific mousechimeras was assessed. Specifically, FACS analysis was carried out toquantify STAT1 phosphorylation in the mouse PD-L1 positive B16 cellline.

B16 cells were stimulated with various bispecific chimeras for 15minutes at 37° C. in DMEM medium supplemented with 10% FBS. Afterstimulation, cells were fixed by adding 1 volume Fix Buffer I (BDBiosciences) for 10 minutes at 37° C., and permeabilized by resuspensionin 2 volumes Perm III Buffer I (BD Biosciences) for 30 minutes on ice.Samples were stained with an anti-STAT1 pY701 antibody (BD Biosciences)for 20 minutes at 4° C. and analyzed with a FACSCalibur (BD Biosciences)and the CellQuest Pro Version 4.0.2 software (BD Biosciences).

As shown in FIG. 14, B16 cells were stimulated with 100 ng/ml ofanti-mouse Sirp1α VHH/anti-mouse PD-L1 VHH/human IFN Q124R bi-specificchimera, anti-mouse DNAM VHH/anti-mouse PD-L1 VHH/human IFN Q124Rbi-specific chimera of Bcll10 VHH-human Q124R IFN or left unstimulatedfor 15 minutes at 37° C. After fixation and permeabilization, cells werestained for phospho STAT1 and analyzed in FACS. Data clearly illustratethat PD-L1 targeting significantly increased STAT1 phosphorylation bythe bispecific chimeras when compared to the untargeted (Bcll10 VHH)chimera.

Example 7. Human CD8 Targeting of Mono-Specific and Bi-Specific Chimeras

The efficiency of human CD8 targeting of mono-specific human chimeraswas examined by quantification of STAT1 phosphorylation in CD8-positiveand CD8-negative peripheral blood mononuclear cells (PBMCs) by FACS.

A chimera of anti-human CD8 VHH/human IFN R149A (i.e.,pmTW-SIgK-hCD8_R2HCD26 (SEQ ID NO:21)-(GGS)₂₀-hIFNa2_R149A-GGS-(His)₉construct) and anti-human CD8 VHH/human IFN R33A/E120A (i.e.,pmTW-SIgK-hCD8_R2HCD26 (SEQ IDNO:21)-(GGS)₂₀-hIFNa2_R33A/E120A-GGS-(His)₉ construct) were produced inHek293F cells. Cells were grown to a density of 0.6×10⁶ cells per ml inFreestyle medium and transfected with 25K PEI (polyethylenimine)according to standard protocols. Three days after transfection, freshmedium was added to the cultures and cells were grown for two or threeadditional days. Medium was harvested, cells removed by centrifugationand filter-sterilized. Recombinant proteins were purified using Ni Excelresin (GE Healthcare) according to the manufacturer's instructions andimidazole removed from the samples with PD10 columns (GE Healthcare).

PBMCs from buffy coats of healthy donors were isolated using densitygradient centrifugation with Ficoll-Paque (GE Healthcare). Cells werewashed twice with FACS buffer (2% FBS, 1 mM EDTA in PBS) and stainedwith anti-human CD8 APC (clone RPE-T8; BD Pharmingen) for 20 minutes at4° C. After two washes, cells were stimulated with a serial dilution ofCD8-targeting chimeras for 15 minutes at 37° C. After fixation (10minutes, 37° C., Fix Buffer I; BD Biosciences) and permeabilisation (30minutes, on ice, Perm III Buffer I; BD Biosciences) and washing, cellswere stained with anti-STAT1 pY701 Ab (BD Biosciences). Samples wereacquired with a FACSCalibur (BD Biosciences), with the CellQuest ProVersion 4.0.2 software (BD Biosciences).

Isolated PBMCs were stimulated with a serial dilution of CD8-targetingchimeras (anti-human CD8 VHH/human IFN R149A, anti-human CD8 VHH/humanIFN R33A/E120A, or anti-Bcll10 VHH/human IFN R149A) and stained for CD8(APC) and pSTAT1 (PE). Data clearly showed that the biological activityof anti-human CD8 VHH/human IFN R149A and anti-Bcll10 VHH-human IFNR149A were comparable in CD8-negative cells (FIG. 15, panel C), but CD8targeting resulted in a clear and pronounced increase (at least 500fold) in STAT1 phosphorylation by anti-human CD8 VHH/human IFN R149A(FIG. 15, panels A and B). The combined IFN mutations in the anti-humanCD8 VHH/human IFN R33A/E120A chimera completely blocked STAT1phosphorylation and CD8 targeting did not rescue biological activity.This was in contrast to the IFN R149A mutation which maintained STAT1phosphorylation.

The efficiency of human CD8 targeting of bi-specific human chimeras(anti-human CD8 VHH/anti-human PD-L1 VHH/human IFN R149A) was alsoexamined by quantification of STAT1 phosphorylation in CD8-positive andCD8-negative peripheral blood mononuclear cells (PBMCs) in FACS.

Anti-human CD8 VHH/anti-human PD-L1 VHH/human IFN R149A bi-specificchimera (i.e., pmTW-SIgK-hCD8_R2HCD26-(GGS)₁₀-hPD-L1VHH-(GGS)₂₀-hIFNa2_R149A-GGS-(His)₉ construct) and anti-human CD8VHH/anti-human PD-L1 VHH/human IFN R33A/E120A bi-specific chimera (i.e.,pmTW-SIgK-hCD8_R2HCD26-(GGS)₁₀-hPD-L1VHH-(GGS)₂₀-hIFNa2_R33A/E120A-GGS-(His)₉ construct) were produced inHek293F cells. Cells were grown to a density of 0.6×10⁶ cells per ml inFreestyle medium and transfected with 25K PEI (polyethylenimine)according to standard protocols. Three days after transfection, freshmedium was added to the cultures and cells were grown for two or threeadditional days. Medium was harvested, cells removed by centrifugationand filter-sterilized. Recombinant proteins were purified using Ni Excelresin (GE Healthcare) according to the manufacturer's instructions andimidazole from the samples removed with PD10 columns (GE Healthcare).

PBMCs from buffy coats of healthy donors were isolated using densitygradient centrifugation with Ficoll-Paque (GE Healthcare). Cells werewashed twice with FACS buffer (2% FBS, 1 mM EDTA in PBS) and stainedwith anti-human CD8 APC (clone RPE-T8; BD Pharmingen) for 20 minutes at4° C. After two washes, cells were stimulated with a serial dilution ofCD8/PD-L1 bi-specific chimeras for 15 minutes at 37° C. After fixation(10 minutes, 37° C., Fix Buffer I; BD Biosciences) and permeabilisation(30 minutes, on ice, Perm III Buffer I; BD Biosciences) and washing,cells were stained with anti-STAT1 pY701 Ab (BD Biosciences). Sampleswere acquired with a FACSCalibur (BD Biosciences), with the CellQuestPro Version 4.0.2 software (BD Biosciences).

Isolated PBMCs were stimulated with a serial dilution of bi-specificchimeras (anti-human CD8 VHH/anti-human PD-L1 VHH/human IFN R149Abi-specific chimera, anti-human CD8 VHH/anti-human PD-L1 VHH/human IFNR33A/E120E bi-specific chimera, or anti-human Bcll10 VHH/human IFNR149A) and stained for CD8 (APC) and pSTAT1 (PE). Anti-human CD8VHH/anti-human PD-L1 VHH/human IFN R149A bi-specific chimera wasapproximately 100-fold more effective in STAT1 phosphorylation thananti-human Bcll10 VHH/human IFN R149A in CD8-negative cells (panels Dand F), pointing to a PD-L1 targeting effect on PD-L1 expressing cells.The effect of CD8 targeting was clearly illustrated by the difference inpSTAT1 levels in CD8 positive cells (FIG. 15, panel E) versus CD8negative cells (FIG. 15, panel F) upon stimulation by the anti-human CD8VHH/anti-human PD-L1 VHH/human IFN R149A bi-specific chimera. Thecombined human IFN R33A/E120A mutation completely blocked STAT1phosphorylation and CD8 and/or PD-L1 targeting did not rescue biologicalactivity. This was in contrast to the R149A mutation which maintainedSTAT1 phosphorylation.

Example 8. Human PD-L1 Targeting of Mono- and Bi-Specific Chimera

The efficiency of human PD-L1 targeting of (bi-specific) human chimeraswas examined by quantification of STAT1 phosphorylation in the humanPD-L1 positive MDA-MB-321 cell-line by FACS analysis. Chimeras studiedwere anti-human PD-L1 VHH/human IFN R149A; anti-human PD-L1 VHH/humanIFN R33A/E120R; anti-human Clec9A VHH/anti-human PD-L1 VHH/human IFNR149A; anti-human Clec9A VHH/anti-human PD-L1 VHH/human IFN R33A/E120R;and anti-human Bcl10 VHH/human IFN R149A.

MDA-MB-321 cells were stimulated with human chimeras as indicated for 15minutes at 37° C. in DMEM medium supplemented with 10% FBS. Afterstimulation, cells were fixed by adding 1 volume Fix Buffer I (BDBiosciences) for 10 minutes at 37° C., and permeabilized by resuspensionin 2 volumes Perm III Buffer I (BD Biosciences) for 30 minutes on ice.Samples were stained with an anti-STAT1 pY701 Ab (BD Biosciences) for 20at 4° C. and analyzed with a FACSCalibur (BD Biosciences) and theCellQuest Pro Version 4.0.2 software (BD Biosciences).

MDA-MB-321 were stimulated with a serial dilution (100 ng/ml; 1 over 5)of mono- (PD-L1 VHH) and bi-specific (Clec9A and PD-L1 VHHs) chimerasfor 15 minutes at 37° C. After fixation and permeabilization, cells werestained for phospho STAT1 and analyzed in FACS. Data illustrate thatPD-L1 targeting (in mono- or bi-specific format) strongly increasedSTAT1 phosphorylation by chimeras having the IFN R149A mutation, but notthe IFN R33A/E120R mutations. Untargeted (Bcll10 VHH) chimera was unableto signal even at 100 ng/ml. See FIG. 16.

Example 9 Bi-Specific Constructs with scFv Targeting Moieties

Expansion of the bi-specific concept beyond VHHs was undertaken. Ananti-human CD20 scFv/anti-human CD3 scFv/IFN R149A of the followingsequence was generated:

(SEQ ID NO: 367) METDTLLLWVLLLWVPGSTG QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYSLDYWGQGTTLTVSSGSTGGGGSGGGGSGGGGSDIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSN PFTFGSGTKLEINRGGSGGQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRGSTGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDW YFNVWGAGTTVTVSSVDGGSGGSGGSGGSGGSGGSRSGGSGGSGGSGGSG

HHH.

The signal peptide is underlined, scFvCD3 is bold, scFvCD20 is bold andunderlined, hIFNa2(R149A) is shaded and linkers connect the targetingmoieties and signaling agent.

The efficiency of targeting by the human scFV-based bi-specific chimerawas examined by quantification of STAT1 phosphorylation in Daudi (CD20positive), Jurkat (CD3) or Wish (negative control) cells. See FIG. 17.

Cells were stimulated with bi-specific chimeras as indicated for 15minutes at 37° C. in DMEM medium supplemented with 10% FBS. Afterstimulation, cells were fixed by adding 1 volume Fix Buffer I (BDBiosciences) for 10 minutes at 37° C., and permeabilised by resuspensionin 2 volumes Perm III Buffer I (BD Biosciences) for 30 minutes on ice.Samples were stained with an anti-STAT1 pY701 Ab (BD Biosciences) for 20minutes at 4° C. and analyzed with a FACSCalibur (BD Biosciences) andthe CellQuest Pro Version 4.0.2 software (BD Biosciences).

As shown in FIG. 17, Daudi, Jurkat and Wish cells were stimulated with aserial dilution (5000 ng/ml; 1 over 5) of scFv chimera for 15 minutes at37° C. After fixation and permeabilization, cells were stained forphospho STAT1 and analyzed by FACS analysis. Data clearly illustratedthat STAT1 phosphorylation was more efficient in Jurkat (CD3-expressing)and Daudi (CD20-expressing) cells compared to Wish cells due to CD3 andCD20 targeting, respectively.

Example 10. Modelled Human Anti-Tumor Effects

Selection of Human Cord Blood

Stem cells were HLA-type matched with the expression of HLA-A2 by thetumor cells used during the experiments. To that end, only HLA-A2positive cord bloods were selected to proceed with CD34 stem cellpurification. Cells were stained with HLA-A2-FITC (BD Pharmingen) orHLA-ABC-PE (BD Pharmingen), the latter as a positive control. Sampleswere acquired on an Attune Nxt Acoustic Focusing Cytometer (LifeTechnologies).

Purification of CD34⁺ Stem Cells from Human Cord Blood

Viable mononuclear cells from human cord bloods were isolated usingFycoll (Lymphoprep, Stemcell technologies) gradient separation prior toCD34⁺ MACS isolation using direct CD34⁺ progenitor cell isolation kit(Miltenyi). Flow cytometric staining using human CD3-PE (BDPhamringen)/human-CD34-APC (BD Pharmingen) was used to evaluate purityof the isolated stem cells. Samples were acquired on an Attune NxtAcoustic Focusing Cytometer (Life Technologies). Purity of injectedcells reached 95-98%.

Generation of Humanized Mice

Newborn NSG mice (1-2 days of age) were sublethally irradiated with 100cGy prior to intrahepatic delivery of 10⁵ CD34⁺ human stem cells. At 6weeks after CD34 transfer, peripheral blood is analyzed for the presenceof both human and mouse CD45 (both BD) cells to analyze the effect ofengraftment. Samples where acquired on a LSR flow cytometer (BD) andanalyzed by FACS Diva software (BD).

The antitumor potential of chimeras was assessed in tumor-bearinghumanized mice models. Newborn NSG mice (1-2 days of age) weresublethally irradiated with 100 cGy prior to intrahepatic delivery of10⁵ CD34⁺ human stem cells (from HLA-A2 positive cord bloods). At week13 after stem cell transfer mice were s.c. inoculated with 2.5×10⁶ humanRL follicular lymphoma cells. Mice were treated i.p. daily started atday 6 after tumor inoculation with 30 μl of Flt3L protein. Dailyperilesional chimera delivery (30 μg) was started at day 10 after tumorinoculation, when a palpable tumor was reached. Treatment with theanti-human Clec9a VHH/anti-human PDL1 VHH/human IFN-R149A bi-specificchimera resulted in stabilization of the tumor growth, which was evenmore pronounced when combined with Flt3L injections in contrast toinjection of PBS. The graph shows tumor growth as mean±SEM.

Importantly, anti-human Clec9a VHH/anti-human PDL1 VHH/human IFN-R149Abi-specific chimera showed clear antitumor effects on a human tumor (RL)grown in humanized mice (mice with a reconstituted human immune system).See FIG. 18.

Example 11. Dendritic Cell Signaling Induced by Anti-Human Bi-SpecificVHH Chimeras

A dendritic cell pSTAT signaling assay was undertaken. Chimeras studiedwere anti-human Clec9A VHH/anti-human PD-L1 VHH/human IFN R149A andanti-human Clec9A VHH/anti-human PD-L1 VHH/human IFN R33A/E120R. Twodoses of the agents were studied: 100 ng/ml and 500 ng/ml.

Briefly, human PBMCs were isolated from blood obtained from healthydonors. Approximately 120 ml of blood was collected from each donorusing heparin coated tubes (12 tubes). The blood was kept at roomtemperature and processed immediately Briefly, blood was diluted 1:1with DPBS and 25 ml was gently layered onto 15 ml of Lympholyte H. Aftercentrifugation, the mononuclear cell rings were collected and cells werewashed three times with DPBS (PBS Dulbecco's Phosphate Buffered Saline,Wisent, catalog #311-425-LL) and counted. Dendritic cells were enrichedfrom the PBMC population using “DC-enrichment kit” containing acombination of lineage specific monoclonal antibodies in PBS and asuspension of magnetic particles (STEMCELL Technologies Catalogue number19251), according to manufacturer's instructions.

Dendritic cells (DC) were stimulated for 15 minutes in the presence orabsence of test items and controls (PBS) and the level ofphosphorylated-STAT1 (pSTAT1, specifically pY701-STAT1) was determinedin isolated DC cell populations (Lin-(CD14/CD16/CD20/CD56/CD3)/HLA-DR+)by flow cytometry. Post stimulation, cells were fixed (BD Cytofixfixation buffer, BD Bioscience, catalog #554655), then permeabilizedwith Perm buffer II (BD PhosFlow Perm Buffer, BD Bioscience, catalog#558052). Cells were then stained for phosphoSTAT1 and for DC surfacemarkers (Lin−/HLA-DR+) (see table below). Both intra-cellular andsurface staining were performed at the same time. Flow cytometry anddata acquisition was performed after cell washing with DPBS.

Table showing list of antibodies for flow cytometry staining

Marker/ Supplier- Product Fluoro- Catalog Name chrome Clone PurposeNumber pSTAT1 AlexaFluor647 4a phospho- BD-562070 STAT1 Anti-human PEUCHT1 T cells marker BD-561809 CD3 Lineage depletion Anti-human PE M5E2Monocytes BD-555398 CD14 markers Lineage depletion anti-human PE  873.1NK, BD-561313 CD16 Neutrophils, Monocytes marker Lineage depletionanti-human PE HIB19 B cells marker BD-555413 CD19 Lineage depletionanti-human PE 8159 NK cells marker BD-555516 CD56 Lineage depletionAnti-human FITC TU36 MHC II marker BD-555560 HLA-DR DC discriminationAnti-human BV421 B-Ly6 DC BD-562561 CD11c discrimination LIVE/DEAD AquaN/Ap Viability dye ThermoFisher- Fixable L34957 Aqua Dead Cell StainNormal N/Ap N/Ap Fc receptor ThermoFisher- mouse IgG blocker 10400CBlocking agent

FIG. 19 shows the data, expressed as a fold change of the percentage ofpSTATf⁺ dendritic cells.

This study clearly shows that a dual (bi-specific) human CLEC9A/PD-L1antigen-targeting construct comprising an IFN signaling agent whoseactivity is recoverable upon cell targeting (IFN R149A) promotes IFNsignaling in human dendritic cells (as determined by pSTAT1 induction).In contrast, no IFN signaling activation is observed with a bi-specificCLEC9A/PD-L1 antigen-targeting construct that incorporates an IFNsignaling agent whose activity is not recoverable (IFN R33A/E120R).Thus, as observed for comparable IFN fusion constructs targeting mouseCLEC9A and PD-L1 antigens, targeting IFN to human dendritic cells usinga targeting moiety directed at human CLEC9A antigen results intriggering of a pronounced IFN signal transduction.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

1.-34. (canceled)
 35. A chimeric protein comprising: (a) a modifiedsignaling agent, wherein the modified signaling agent is a mutant humaninterferon alpha 2 (IFNα2) comprising an amino acid sequence having atleast 95% identity with SEQ ID NO: 336 and having a mutation at positionR149; (b) a first targeting moiety comprising a first recombinant heavychain-only (VHH) antibody or single-chain variable fragment (scFv),wherein the first targeting moiety specifically binds to a first antigenor receptor of interest; and (c) a second targeting moiety comprising asecond recombinant heavy chain-only (VHH) antibody or single-chainvariable fragment (scFv), wherein the second targeting moietyspecifically binds to a second antigen or receptor of interest; whereinthe mutant human IFNα2 has a reduced affinity or activity for itsreceptor relative to the wild type human IFNα2; and wherein the reducedaffinity or activity of the mutant human IFNα2 for its receptor isrestorable by one or more of the targeting moieties.
 36. The chimericprotein of claim 35, wherein the first targeting moiety is attached tothe second targeting moiety and the second targeting moiety is attachedto the modified signaling agent.
 37. The chimeric protein of claim 35,wherein both the first and the second targeting moieties are attached tothe modified signaling agent.
 38. The chimeric protein of claim 35,wherein the human IFNα2 further comprises one or more mutations atpositions 144-154.
 39. The chimeric protein of claim 38, wherein the oneor more mutations is at positions M148 or L153.
 40. The chimeric proteinof claim 35, wherein the mutation is R149A.
 41. The chimeric protein ofclaim 39, wherein the mutation is M148A.
 42. The chimeric protein ofclaim 39, wherein the mutation is L153A.
 43. The chimeric protein ofclaim 35, wherein the first or the second antigen or receptor ofinterest is C-type lectin domain family 9 member A (Clec9A), ProgrammedDeath-Ligand 1 (PD-L1), Cluster of Differentiation 3 (CD3), Cluster ofDifferentiation 8 (CD8), Cluster of Differentiation 20 (CD20), SignalRegulatory Protein Alpha (Sirp1α), or DNAX Accessory Molecule (DNAM).44. The chimeric protein of claim 35, wherein the first antigen orreceptor of interest is PD-L1and the second antigen or receptor ofinterest is CD20, Clec9A, CD8, Sirp1α, or DNAM.
 45. The chimeric proteinof claim 35, wherein the first antigen or receptor of interest is Clec9Aand the second antigen or receptor of interest is CD20.
 46. The chimericprotein of claim 35, wherein the first antigen or receptor of interestis PD-L1 and the second antigen or receptor of interest is Clec9A. 47.The chimeric protein of claim 35, wherein the first antigen or receptorof interest is CD20 and the second antigen or receptor of interest isCD3.
 48. The chimeric protein of claim 35, wherein: (a) the firsttargeting moiety functionally modulates the activity of the firstantigen or receptor of interest; (b) the second targeting moietyfunctionally modulates the activity of the second antigen or receptor ofinterest; (c) the first targeting moiety does not functionally modulatethe activity of the first antigen or receptor of interest; or (d) thesecond targeting moiety does not functionally modulate the activity ofthe second antigen or receptor of interest.
 49. The chimeric protein ofclaim 35, wherein the first targeting moiety, the second targetingmoiety, or both comprise a VHH, a humanized VHH, or a camelized VHH. 50.The chimeric protein of claim 35, wherein the first targeting moiety,the second targeting moiety, or both comprise a scFv.
 51. A method fortreating cancer, comprising administering to a subject in need thereofan effective amount of a chimeric protein comprising: (a) a modifiedsignaling agent, wherein the modified signaling agent is a mutant humaninterferon alpha 2 (IFNα2) comprising an amino acid sequence having atleast 95% identity with SEQ ID NO: 336 and having a mutation at positionR149; (b) a first targeting moiety comprising a first recombinant heavychain-only (VHH) antibody or single-chain variable fragment (scFv),wherein the first targeting moiety specifically binds to a first antigenor receptor of interest; and (c) a second targeting moiety comprising asecond recombinant heavy chain-only (VHH) antibody or single-chainvariable fragment (scFv), wherein the second targeting moietyspecifically binds to a second antigen or receptor of interest; whereinthe mutant human IFNα2 has a reduced affinity or activity for itsreceptor relative to the wild type human IFNα2; and wherein the reducedaffinity or activity of the mutant human IFNα2 for its receptor isrestorable by one or more of the targeting moieties.
 52. The method ofclaim 51, wherein the first targeting moiety is attached to the secondtargeting moiety and the second targeting moiety is attached to themodified signaling agent.
 53. The method of claim 51, wherein both thefirst and the second targeting moieties are attached to the modifiedsignaling agent.
 54. The method of claim 51, wherein the human IFNα2further comprises one or more mutations at positions 144-154.
 55. Themethod of claim 54, wherein the one or more mutations is at positionsM148 or L153.
 56. The method of claim 51, wherein the mutation is R149A.57. The method claim 55, wherein the mutation is M148A.
 58. The methodclaim 55, wherein the mutation is L153A.
 59. The method of claim 51,wherein the first or the second antigen or receptor of interest isC-type lectin domain family 9 member A (Clec9A), Programmed Death-Ligand1 (PD-L1), Cluster of Differentiation 3 (CD3), Cluster ofDifferentiation 8 (CD8), Cluster of Differentiation 20 (CD20), SignalRegulatory Protein Alpha (Sirp1α), or DNAX Accessory Molecule (DNAM).60. The method of claim 51, wherein the first antigen or receptor ofinterest is PD-L1 and the second antigen or receptor of interest isCD20, Clec9A, CD8, Sirp1α, or DNAM.
 61. The method of claim 51, whereinthe first antigen or receptor of interest is Clec9A and the secondantigen or receptor of interest is CD20.
 62. The method of claim 51,wherein the first antigen or receptor of interest is PD-L1 and thesecond antigen or receptor of interest is Clec9A.
 63. The method ofclaim 51, wherein the first antigen or receptor of interest is CD20 andthe second antigen or receptor of interest is CD3.
 64. The method ofclaim 51, wherein: (a) the first targeting moiety functionally modulatesthe activity of the first antigen or receptor of interest; (b) thesecond targeting moiety functionally modulates the activity of thesecond antigen or receptor of interest; (c) the first targeting moietydoes not functionally modulate the activity of the first antigen orreceptor of interest; or (d) the second targeting moiety does notfunctionally modulate the activity of the second antigen or receptor ofinterest.
 65. The method of claim 51, wherein the first targetingmoiety, the second targeting moiety, or both comprise a VHH, a humanizedVHH, or a camelized VHH.
 66. The method of claim 51, wherein the firsttargeting moiety, the second targeting moiety, or both comprise a scFv.